Signal boost cascade assay

ABSTRACT

The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods provide signal boost upon detection of target nucleic acids of interest in less than one minute and in some instances instantaneously at ambient temperatures down to 16° C. or less, without amplification of the target nucleic acids yet allowing for massive multiplexing, high accuracy and minimal non-specific signal generation.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 18/078,821, filed 9Dec. 2022, which claims priority to U.S. Ser. No. 63/289,112, filed 13Dec. 2021; U.S. Ser. No. 63/359,183, filed 7 Jul. 2022; U.S. Ser. No.63/395,394, filed 5 Aug. 2022; and U.S. Ser. No. 63/397,785, filed 12Aug. 2022.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

Submitted herewith is an electronically filed sequence listing viaEFS-Web a Sequence Listing XML, entitled “LS004US1_seqlist_20221201”,created 1 Dec. 2022, which is 1,227,000 bytes in size. The sequencelisting is part of the specification of this specification and isincorporated by reference in its entirety.

PETITION UNDER 37 CFR 1.84(a)(2)

This patent application contains at least one drawing executed in color.The color drawings are necessary as the only practical medium by whichaspects of the claimed subject matter may be accurately conveyed. Theclaimed invention relates to variant proteins that alter the active sitethereof and the color drawings are necessary to easily discern thestructural difference between variants. As the color drawings are beingfiled electronically via EFS-Web, only one set of the drawings isrequired.

FIELD OF THE INVENTION

The present disclosure relates to compositions of matter and assaymethods used to detect one or more target nucleic acids of interest in asample. The compositions and methods provide a signal boost upondetection of target nucleic acids of interest in less than one minuteand at ambient temperatures down to 16° C. or less.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Rapid and accurate identification of, e.g., infectious agents, microbecontamination, variant nucleic acid sequences that indicate the presentof diseases such as cancer or contamination by heterologous sources isimportant in order to select correct treatment; identify tainted food,pharmaceuticals, cosmetics and other commercial goods; and to monitorthe environment including identification of biothreats. Classic PCR andnucleic acid-guided nuclease or CRISPR (clustered regularly interspacedshort palindromic repeats) detection methods rely on pre-amplificationof target nucleic acids of interest to enhance detection sensitivity.However, amplification increases time to detection and may cause changesto the relative proportion of nucleic acids in samples that, in turn,lead to artifacts or inaccurate results. Improved technologies thatallow very rapid and accurate detection of nucleic acids are thereforeneeded for timely diagnosis and treatment of disease, to identify toxinsin consumables and the environment, as well as in other applications.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure provides compositions of matter and assay methodsto detect target nucleic acids of interest. The “nucleic acid-guidednuclease cascade assays” or “signal boost cascade assays” or “cascadeassays” described herein comprise two different ribonucleoproteincomplexes and either blocked nucleic acid molecules or blocked primermolecules. The blocked nucleic acid molecules or blocked primermolecules keep one of the ribonucleoprotein complexes “locked” unlessand until a target nucleic acid of interest activates the otherribonucleoprotein complex. The present nucleic acid-guided nucleasecascade assay can detect one or more target nucleic acids of interest(e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower) limits in lessthan one minute and in some embodiments virtually instantaneouslywithout the need for amplifying the target nucleic acid(s) of interest,thereby avoiding the drawbacks of multiplex DNA amplification, such asprimer-dimerization. Further, the cascade assay prevents “leakiness”that can lead to non-specific signal generation resulting in falsepositives by preventing unwinding of the blocked nucleic acid moleculesor blocked primer molecules (double-stranded molecules); thus, thecascade assay is quantitative in addition to being rapid. A particularlyadvantageous feature of the cascade assay is that, with the exception ofthe gRNA in RNP1, the cascade assay components are the same in eachassay no matter what target nucleic acid(s) of interest is beingdetected; moreover, the gRNA in the RNP1 is easily reprogrammed usingtraditional guide design methods.

The present disclosure is related first, to the instantaneous cascadeassay, and second, to three modalities for preventing any “leakiness” inthe cascade assay leading to false positives. The three modalitiesenhance the cascade assay and are in addition to using blocked nucleicacid molecules or blocked primer molecules in the cascade assay.

A first embodiment provides a method for identifying a target nucleicacid of interest in a sample in one minute or less at 16° C. or morecomprising the steps of: providing a reaction mixture comprising: firstribonucleoprotein complexes (RNP1s) each comprising a first nucleicacid-guided nuclease and a first gRNA, wherein the first gRNA comprisesa sequence complementary to the target nucleic acid of interest; andwherein binding of the RNP1 complex to the target nucleic acid ofinterest activates cis-cleavage and trans-cleavage activity of the firstnucleic acid-guided nuclease; second ribonucleoprotein complexes (RNP2s)comprising a second nucleic acid-guided nuclease and a second gRNA thatis not complementary to the target nucleic acid of interest; wherein thesecond nucleic acid-guided nuclease optionally comprises a variantnuclease engineered such that single stranded DNA is cleaved faster thandouble stranded DNA is cleaved, wherein the variant nuclease comprisesat least one mutation to the domains that interact with the PAM regionor surrounding sequences on blocked nucleic acid molecules, and whereinthe variant nuclease exhibits both cis- and trans-cleavage activity; aplurality of the blocked nucleic acid molecules comprising a sequencecorresponding to the second gRNA, wherein the blocked nucleic acidmolecules comprise: a first region recognized by the RNP2 complex; oneor more second regions not complementary to the first region forming atleast one loop; one or more third regions complementary to andhybridized to the first region forming at least one clamp, wherein theplurality of blocked nucleic acid molecules and the RNP2s optionally areat a concentration ratio where the blocked nucleic acid molecules are atan equal or higher molar concentration than the RNP2s in the reactionmixture, wherein the blocked nucleic acid molecules optionally eachcomprise at least one bulky modification, and wherein the reactionmixture comprises at least one of a variant nuclease, the concentrationratio of the blocked nucleic acid molecules at a higher molarconcentration than the molar concentration of RNP2s in the reactionmixture, and/or the blocked nucleic acid molecules comprise at least onebulky modification; contacting the reaction mixture with the sampleunder conditions that allow the target nucleic acid of interest in thesample to bind to RNP1, wherein upon binding of the target nucleic acidof interest RNP1 becomes active initiating trans-cleavage of at leastone of the plurality of blocked nucleic acid molecules thereby producingat least one unblocked nucleic acid molecule, and wherein the at leastone unblocked nucleic acid molecule binds to RNP2 initiatingtrans-cleavage of at least one further blocked nucleic acid molecule;and detecting the cleavage products, thereby detecting the targetnucleic acid of interest in the sample in one minute or less.

An additional embodiment provides a method for identifying a targetnucleic acid of interest in a sample in one minute or less at 16° C. ormore comprising the steps of: providing a reaction mixture comprising:first ribonucleoprotein complexes (RNP1s), wherein the RNP1s comprise afirst nucleic acid-guided nuclease and a first guide RNA (gRNA); whereinthe first gRNA comprises a sequence complementary to the nucleic acidtarget of interest, and wherein the first nucleic acid-guided nucleaseexhibits both cis-cleavage activity and trans-cleavage activity; secondribonucleoprotein complexes (RNP2s) comprising a second nucleicacid-guided nuclease and a second gRNA that is not complementary to thetarget nucleic acid of interest; wherein the second nucleic acid-guidednuclease optionally comprises a variant nuclease engineered such thatsingle stranded DNA is cleaved faster than double stranded DNA iscleaved, wherein the variant nuclease comprises at least one mutation tothe domains that interact with the PAM region or surrounding sequenceson a synthesized activating molecule, and wherein the variant nucleaseexhibits both cis- and trans-cleavage activity; a plurality of templatemolecules comprising sequence homology to the second gRNA; a pluralityof the blocked primer molecules comprising a sequence complementary tothe template molecules, wherein the blocked primer molecules cannot beextended by a polymerase, and wherein the blocked primer moleculescomprise: a first region recognized by the RNP2; one or more secondregions not complementary to the first region forming at least one loop;and one or more third regions complementary to and hybridized to thefirst region forming at least one clamp, wherein the plurality ofblocked primer molecules and the RNP2s optionally are at a concentrationratio where the blocked nucleic acid molecules are at a higher molarconcentration than the RNP2s in the reaction mixture, wherein theblocked primer molecules each optionally comprise at least one bulkymodification, and wherein the reaction mixture comprises at least one ofa variant nuclease, a concentration ratio where the blocked nucleic acidmolecules are at a higher molar concentration than the RNP2s in thereaction mixture, and/or the blocked nucleic acid molecules comprisingat least one bulky modification; and a polymerase and a plurality ofnucleotides; contacting the reaction mixture with the sample underconditions that allow nucleic acid targets of interest in the sample tobind to RNP1, wherein: upon binding of the nucleic acid targets ofinterest to the RNP1, the RNP1 becomes active trans-cleaving at leastone of the blocked primer molecules, thereby producing at least oneunblocked primer molecule that can be extended by the polymerase; the atleast one unblocked primer molecule binds to one of the templatemolecules and is extended by the polymerase and nucleotides to form atleast one synthesized activating molecule having a sequencecomplementary to the second gRNA; and the at least one synthesizedactivating molecule binds to the second gRNA, and RNP2 becomes activecleaving at least one further blocked primer molecule and at least onereporter moiety in a cascade; allowing the cascade to continue; anddetecting the unblocked primer molecules, thereby detecting the targetnucleic acid of interest in the sample in one minute or less.

Aspects of the embodiments of the methods for identifying a targetnucleic acid of interest in a sample in one minute or less can besubstituted for any assay for identifying target nucleic acids; forexample, for detecting human pathogens; animal pathogens; diseasebiomarkers; pathogens in laboratories, food processing facilities,hospitals, and in the environment, including bioterrorism applications(see the exemplary organisms listed in Tables 1, 2, 3, 5 and 6 and theexemplary human biomarkers listed in Table 4). Suitable samples fortesting include any environmental sample, such as air, water, soil,surface, food, clinical sites and products, industrial sites andproducts, pharmaceuticals, medical devices, nutraceuticals, cosmetics,personal care products, agricultural equipment and sites, and commercialsamples, and any biological sample obtained from an organism or a partthereof, such as a plant, animal (including humans), or microbe.

There is also provided in an embodiment a method of detecting a targetnucleic acid molecule in a sample in a cascade reaction comprising thesteps of: (a) providing a reaction mixture comprising: (i) a firstribonucleoprotein complex (RNP1) comprising a first nucleic acid-guidednuclease and a first guide RNA (gRNA) comprising a sequencecomplementary to a target nucleic acid molecule; (ii) a secondribonucleoprotein complex (RNP2) comprising a second nucleic acid-guidednuclease and a second gRNA that is not complementary to the targetnucleic acid molecule; and (iii) a plurality of blocked nucleic acidmolecules comprising a sequence complementary to the second guide RNA,(b) contacting the target nucleic acid molecule with the reactionmixture under conditions that, relative to a control reaction, reducethe probability of R-loop formation between the second gRNA and theplurality of blocked nucleic acid molecules, wherein: (i) upon bindingof the target nucleic acid molecule, the RNP1 becomes active wherein thefirst nucleic acid-guided nuclease cleaves at least one of the blockednucleic acid molecules, thereby producing at least one unblocked nucleicacid molecule; and (ii) at least one unblocked nucleic acid moleculebinds to the second gRNA, and the RNP2 becomes active wherein the secondnucleic acid-guided nuclease cleaves at least one further blockednucleic acid molecule; and (c) detecting the cleavage products of step(b), thereby detecting the target nucleic acid molecule in the sample.

There is also provided a second embodiment comprising a method ofincreasing the efficiency, reducing the background, increasing thesignal-to-noise ratio, reducing cis-cleavage of blocked nucleic acidmolecules and preventing unwinding of the second ribonucleoproteincomplex (RNP2) in a cascade reaction comprising: (a) a reaction mixturecomprising: (i) a first ribonucleoprotein complex (RNP1) comprising afirst nucleic acid-guided nuclease and a first guide RNA (gRNA)comprising a sequence complementary to a target nucleic acid molecule;(ii) the RNP2 comprising a second nucleic acid-guided nuclease and asecond gRNA that is not complementary to the target nucleic acidmolecule; and (iii) a plurality of blocked nucleic acid moleculescomprising a sequence complementary to the second guide RNA, and (b) thetarget nucleic acid molecule comprising a sequence complementary to thefirst gRNA; and the method comprising the step of initiating the cascadereaction by contacting (a) and (b) under conditions that reduce theprobability of R-loop formation between the blocked nucleic acidmolecules and the second gRNA, thereby reducing increasing theefficiency, reducing the background, increasing the signal-to-noiseratio, reducing cis-cleavage of blocked nucleic acid molecules andpreventing unwinding of the RNP2 relative to a control reaction.

There is also provided in a third embodiment a method of increasing thesignal-to-noise ratio in a cascade reaction comprising the steps of: (a)providing a reaction mixture comprising: (i) a first ribonucleoproteincomplex (RNP1) comprising a first nucleic acid-guided nuclease and afirst guide RNA (gRNA) comprising a sequence complementary to a targetnucleic acid molecule; (ii) a second ribonucleoprotein complex (RNP2)comprising a second nucleic acid-guided nuclease and a second gRNA thatis not complementary to the target nucleic acid molecule; and (iii) aplurality of blocked nucleic acid molecules comprising a sequencecomplementary to the second guide RNA, (b) initiating the cascadereaction by contacting the target nucleic acid molecule with thereaction mixture under conditions that reduce the probability of R-loopformation between the second gRNA and the plurality of blocked nucleicacid molecules, thereby increasing the signal-to-noise ratio in thecascade reaction relative to a control reaction, wherein: (i) uponbinding of the target nucleic acid molecule, the RNP1 becomes activecleaving at least one of the blocked nucleic acid molecules, therebyproducing at least one unblocked nucleic acid molecule; and (ii) theleast one unblocked nucleic acid molecule binds to the second gRNA, andthe RNP2 becomes active cleaving at least one further blocked nucleicacid molecule; and (c) detecting the cleavage products of the cascadereaction in step (b); and (d) determining the signal-to-noise ratio ofthe cascade reactions in step (b).

A fourth embodiment provides a method of increasing the efficiency,reducing the background, increasing the signal-to-noise ratio, reducingcis-cleavage of blocked nucleic acid molecules and preventing unwindingof a second ribonucleoprotein complex (RNP2) in a cascade reactioncomprising the steps of: (a) providing a reaction mixture comprising: afirst ribonucleoprotein complex (RNP1) comprising a first nucleicacid-guided nuclease and a first guide RNA (gRNA) comprising a sequencecomplementary to a target nucleic acid molecule; the RNP2 comprising asecond nucleic acid-guided nuclease and a second gRNA that is notcomplementary to the target nucleic acid molecule; and a plurality ofblocked nucleic acid molecules comprising a sequence complementary tothe second guide RNA, (b) initiating the cascade reaction by contactingthe target nucleic acid molecule with the reaction mixture underconditions that reduce the probability of R-loop formation between thesecond gRNA and the plurality of blocked nucleic acid molecules, therebyincreasing the efficiency, reducing the background, increasing thesignal-to-noise ratio, reducing cis-cleavage of blocked nucleic acidmolecules and preventing unwinding of the RNP2 in the cascade reactionrelative to a control reaction.

In some aspects of these embodiments, the conditions that reduce R-loopformation comprise one or more of the steps of: 1) providing a molarconcentration of blocked nucleic acid molecules that exceeds the molarconcentration of ribonucleoprotein complexes; 2) engineering the nucleicacid-guided nuclease used in the ribonucleoprotein complex to result ina variant nucleic acid-guided nuclease such that single stranded DNA iscleaved faster than double stranded DNA is cleaved; and/or 3)engineering the blocked nucleic acid molecules to include bulkymodifications of a size of about 1 nm or less.

Another embodiment provides a method for preventing unwinding of blockednucleic acid molecules in the presence of an RNP in a cascade reactioncomprising the steps of: providing blocked nucleic acid molecules;providing ribonucleoprotein complexes comprising a nucleic acid-guidednuclease that exhibits both cis- and trans-cleavage activity uponactivation and a gRNA that recognizes an unblocked nucleic acid moleculeresulting from trans-cleavage of the blocked nucleic acid molecules; andproviding a molar concentration of the blocked nucleic acid moleculesthat exceeds the molar concentration of ribonucleoprotein complexes;engineering the nucleic acid-guided nuclease used in theribonucleoprotein complex to result in a variant nucleic acid-guidednuclease such that single stranded DNA is cleaved faster than doublestranded DNA is cleaved; and/or 3) engineering the blocked nucleic acidmolecules to include bulky modifications of a size of about 1 nm or lessthereby preventing unwinding of the blocked nucleic acid molecules inthe cascade reaction.

In some aspects of the aforementioned embodiments, the blocked nucleicacid molecules are blocked primer molecules.

In a further embodiment, there is provided a method for preventingunwinding of blocked nucleic acid molecules or blocked primer moleculesin the presence of an RNP comprising the steps of: providing blockednucleic acid molecules or blocked primer molecules; providingribonucleoprotein complexes comprising a nucleic acid-guided nucleasethat exhibits both cis- and trans-cleavage activity upon activation anda gRNA that recognizes an unblocked nucleic acid molecule or anunblocked primer molecule resulting from trans-cleavage of the blockednucleic acid molecule or blocked primer molecule; and providing a molarconcentration of blocked nucleic acid molecules that exceeds the molarconcentration of ribonucleoprotein complexes; engineering the nucleicacid-guided nuclease used in the ribonucleoprotein complex to result ina variant nucleic acid-guided nuclease such that single stranded DNA iscleaved times faster than double stranded DNA is cleaved; and/or 3)engineering the blocked nucleic acid molecules to include bulkymodifications of a size of about 1 nm or less.

Other embodiments provide a method for detecting target nucleic acidmolecules in a sample in less than one minute without amplifying thetarget nucleic acid molecules; and instantaneously detecting targetnucleic acid molecules in a sample without amplifying the target nucleicacid molecules.

In some aspects of the methods, the reaction mixture is provided at 16°C., and in some aspects, the reaction mixture is provided at 17° C., 18°C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27°C., 28° C., 29° C., or 30° C. or higher.

Other embodiments provide reaction mixtures for identifying a targetnucleic acid of interest in a sample in one minute or less comprising:first ribonucleoprotein (RNP1) complexes (RNP1s) each comprising a firstnucleic acid-guided nuclease and a first gRNA, wherein the first gRNAcomprises a sequence complementary to the target nucleic acid ofinterest; and wherein binding of the RNP1 complex to the target nucleicacid of interest activates cis-cleavage and trans-cleavage activity ofthe first nucleic acid-guided nuclease; second ribonucleoproteincomplexes (RNP2s) comprising a second nucleic acid-guided nuclease and asecond gRNA that is not complementary to the target nucleic acid ofinterest; wherein the second nucleic acid-guided nuclease optionallycomprises a variant nuclease engineered such that single stranded DNA iscleaved faster than double stranded DNA is cleaved, wherein the variantnuclease comprises at least one mutation to the domains that interactwith the PAM region or surrounding sequences on the blocked nucleic acidmolecules, and wherein the variant nuclease exhibits both cis- andtrans-cleavage activity; and a plurality of the blocked nucleic acidmolecules comprising a sequence corresponding to the second gRNA,wherein the blocked nucleic acid molecules comprise: a first regionrecognized by the RNP2 complex; one or more second regions notcomplementary to the first region forming at least one loop; one or morethird regions complementary to and hybridized to the first regionforming at least one clamp, and wherein the blocked nucleic acidmolecules optionally each comprise at least one bulky modification,wherein the plurality of blocked nucleic acid molecules and the RNP2soptionally are at a concentration ratio where blocked nucleic acidmolecules are at a higher molar concentration than the RNP2s in thereaction mixture, and wherein the reaction mixture comprises at leastone of a variant nuclease, a concentration ratio where blocked nucleicacid molecules are at a higher molar concentration than the RNP2s in thereaction mixture, and/or blocked nucleic acid molecules comprising atleast one bulky modification.

Also provided is a reaction mixture for identifying a target nucleicacid of interest in a sample in one minute or less comprising: firstribonucleoprotein complexes (RNP1s), wherein the RNP1s comprise a firstnucleic acid-guided nuclease and a first guide RNA (gRNA); wherein thefirst gRNA comprises a sequence complementary to the nucleic acid targetof interest, and wherein the first nucleic acid-guided nuclease exhibitsboth cis-cleavage activity and trans-cleavage activity; secondribonucleoprotein complexes (RNP2s) comprising a second nucleicacid-guided nuclease and a second gRNA that is not complementary to thetarget nucleic acid of interest; wherein the second nucleic acid-guidednuclease optionally comprises a variant nuclease engineered such thatsingle stranded DNA is cleaved faster than double stranded DNA iscleaved, wherein the variant nuclease comprises at least one mutation tothe domains that interact with the PAM region or surrounding sequenceson synthesized activating molecules, and wherein the variant nucleaseexhibits both cis- and trans-cleavage activity; a plurality of templatemolecules comprising sequence homology to the second gRNA; a pluralityof the blocked primer molecules comprising a sequence complementary tothe template molecules, wherein the blocked primer molecules cannot beextended by a polymerase, and wherein the blocked primer moleculescomprise: a first region recognized by the RNP2; one or more secondregions not complementary to the first region forming at least one loop;and one or more third regions complementary to and hybridized to thefirst region forming at least one clamp, wherein the blocked primermolecules optionally each comprise at least one bulky modification andwherein the plurality of blocked primer molecules and the RNP2soptionally are at a concentration ratio where blocked nucleic acidmolecules are at a higher molar concentration than the RNP2s in thereaction mixture, and wherein the reaction mixture comprises at leastone of a variant nuclease, at a concentration ratio where blockednucleic acid molecules are at a higher molar concentration than theRNP2s in the reaction mixture, and/or blocked nucleic acid moleculescomprising at least one bulky modification; and a polymerase and aplurality of nucleotides.

Further provided is a composition of matter comprising:ribonucleoprotein complexes (RNPs) comprising a nucleic acid-guidednuclease and a gRNA that is not complementary to the target nucleic acidof interest; wherein the nucleic acid-guided nuclease optionallycomprises a variant nuclease engineered such that single stranded DNA iscleaved faster than double stranded DNA is cleaved, wherein the variantnuclease comprises at least one mutation to the domains that interactwith the PAM region or surrounding sequences on the blocked nucleic acidmolecules, and wherein the variant nuclease exhibits both cis- andtrans-cleavage activity; and a plurality of the blocked nucleic acidmolecules comprising a sequence corresponding to the gRNA, wherein theblocked nucleic acid molecules comprise: a first region recognized bythe RNP complex; one or more second regions not complementary to thefirst region forming at least one loop; one or more third regionscomplementary to and hybridized to the first region forming at least oneclamp, wherein the blocked nucleic acid molecules each comprise at leastone bulky modification, wherein the blocked nucleic acid moleculesoptionally each comprise at least one bulky modification, and whereinthe plurality of blocked nucleic acid molecules and the RNP2s optionallyare at a concentration ratio where the blocked nucleic acid moleculesare at a higher molar concentration than the RNP2s in the reactionmixture, and wherein the composition comprises at least one of a variantnuclease, a concentration ratio where the blocked nucleic acid moleculesare at a higher molar concentration than the RNP2s in the reactionmixture, and/or blocked nucleic acid molecules comprising at least onebulky modification; and a polymerase and a plurality of nucleotides.

Additionally provided is a composition of matter comprising:ribonucleoprotein complexes (RNPs) comprising a nucleic acid-guidednuclease and a gRNA that is not complementary to the target nucleic acidof interest; wherein the second nucleic acid-guided nuclease optionallycomprises a variant nuclease engineered such that single stranded DNA iscleaved faster than double stranded DNA is cleaved, wherein the variantnuclease comprises at least one mutation to the domains that interactwith the PAM region or surrounding sequences on the blocked nucleic acidmolecules, and wherein the variant nuclease exhibits both cis- andtrans-cleavage activity; a plurality of template molecules comprisingsequence homology to the gRNA; and a plurality of the blocked primermolecules comprising a sequence complementary to the template molecules,wherein the blocked primer molecules cannot be extended by a polymerase,and wherein the blocked primer molecules comprise: a first regionrecognized by the RNP2; one or more second regions not complementary tothe first region forming at least one loop; and one or more thirdregions complementary to and hybridized to the first region forming atleast one clamp, wherein the blocked primer molecules optionally eachcomprise at least one bulky modification, and wherein the plurality ofblocked primer molecules and the RNPs optionally are at a concentrationwhere the blocked nucleic acid molecules are at a molar concentrationequal to or greater than the molar concentration of the RNPs in thereaction mixture, and wherein the composition comprises at least one ofa variant nuclease, a concentration ratio where blocked nucleic acidmolecules are at a higher molar concentration than the RNP2s in thereaction mixture, and/or blocked nucleic acid molecules comprising atleast one bulky modification; and a polymerase and a plurality ofnucleotides.

In some aspects of these embodiments, the reaction mixture furthercomprises reporter moieties, wherein the reporter moieties produce adetectable signal upon trans-cleavage activity by the RNP2 to identifythe presence of one or more nucleic acid targets of interest in thesample. In some aspects, the reporter moieties are not coupled to theblocked primer molecules, and wherein upon cleavage by RNP2, a signalfrom the reporter moiety is detected; yet in other aspects, the reportermoieties are coupled to the blocked primer molecules, and wherein uponcleavage by RNP2, a signal from the reporter moiety is detected.

In some aspects of all embodiments comprising bulky modifications, thebulky modifications are about 1 nm in size, and in some aspects, thebulky modifications are about 0.9 nm, 0.8 nm, 0.7 nm, 0.6 nm, 0.5 nm,0.4 nm, 0.3 nm, 0.2 nm, or 0.1 nm in size. In some aspects, the bulkymodifications are about 0.9 nm, 0.8 nm, 0.7 nm, 0.6 nm, 0.5 nm, 0.4 nm,0.3 nm, 0.2 nm, or 0.1 nm in size. In some aspects, the blocked nucleicacid molecules include bulky modifications and wherein there are twobulky modifications with one bulky modification located on the 5′ end ofthe blocked nucleic acid molecule and one bulky modification located onthe 3′ end of the blocked nucleic acid molecule, and where the 5′ and 3′ends comprising the two bulky modifications are less than 11 nm from oneanother. In other aspects, the bulky modification is on a 5′ end ofblocked nucleic acid molecules and may be selected from the group of 5′Fam (6-fluorescein amidite); Black Hole Quencher-1-5; biotin TEG (15atom triethylene glycol spacer); biotin-5; and cholesterol TEG (15 atomtriethylene glycol spacer). In other aspects, the bulky modification ison a 3′ end of the blocked nucleic acid molecules and may be selectedfrom the group of Black Hole Quencher-1-3; biotin-3; and TAMRA-3′(carboxytetramethylrhodamine). In some aspects, a bulky modification isbetween two internal nucleic acid residues of the blocked nucleic acidmolecules and may be selected from the group of Cy3 internal and Cy5,and in some aspects, the bulky modification is an internal nucleotidebase modification and may be selected from the group of biotindeoxythymidine dT; disthiobiotin NHS; and fluorescein dT.

In some aspects of these embodiments, the blocked nucleic acid moleculesor blocked primer molecules comprise a structure represented by any oneof Formulas I-IV, wherein Formulas I-IV are in the 5′-to-3′ direction:

(a) A-(B-L)J-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;    -   B is 4-12 nucleotides in length;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10;    -   C is 4-15 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then A-(B-L)J-C and T-D are separate nucleic acid        strands;    -   T is 17-135 nucleotides in length and comprises at least 50%        sequence complementarity to B and C; and    -   D is 0-10 nucleotides in length and comprises at least 50%        sequence complementarity to A;

(b) D-T-T′-C-(L-B)J-A  (Formula II);

-   -   wherein D is 0-10 nucleotides in length;    -   T-T′ is 17-135 nucleotides in length;    -   T′ is 1-10 nucleotides in length and does not hybridize with T;    -   C is 4-15 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length and does not hybridize with T;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   J is an integer between 1 and 10;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;

(c) T-D-M-A-(B-L)J-C  (Formula III);

wherein T is 17-135 nucleotides in length;

-   -   D is 0-10 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then T-D and A-(B-L)J-C are separate nucleic acid        strands;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10; and    -   C is 4-15 nucleotides in length; or

(d) T-D-M-A-Lp-C  (Formula IV);

-   -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-15 nucleotides in length;    -   M is 1-25 nucleotides in length;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D; and    -   L is 3-25 nucleotides in length;    -   p is 0 or 1;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T.

In some aspects, (a) T of Formula I comprises at least 80% sequencecomplementarity to B and C; (b) D of Formula I comprises at least 80%sequence complementarity to A; (c) C of Formula II comprises at least80% sequence complementarity to T; (d) B of Formula II comprises atleast 80% sequence complementarity to T; (e) A of Formula II comprisesat least 80% sequence complementarity to D; (f) A of Formula IIIcomprises at least 80% sequence complementarity to D; (g) B of FormularIII comprises at least 80% sequence complementarity to T; (h) A ofFormula IV comprises at least 80% sequence complementarity to D; and/or(i) C of Formula IV comprises at least 80% sequence complementarity toT.

In some aspects, the variant nucleic acid-guided nuclease is a Type Vvariant nucleic acid-guided nuclease. In some aspects, the one or bothof the RNP1 and the RNP2 comprise a nucleic acid-guided nucleaseselected from Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14,Cas12h, Cas12i, Cas12j, Cas13a, or Cas13b.

In some aspects of the embodiments that comprise a variant nucleicacid-guided nuclease, the variant nucleic acid-guided nuclease comprisesat least one mutation to the domains that interact with the PAM regionor surrounding sequences on the blocked nucleic acid molecules whereinthe mutation is selected from mutations to amino acid residues K538,Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acidresidues in orthologs. In some embodiments, there are at least twomutations to the domains that interact with the PAM region orsurrounding sequences on the blocked nucleic acid molecules selectedfrom mutations to amino acid residues K538, Y542 and K595 in relation toSEQ ID NO:1 and equivalent amino acid residues in orthologs and in otheraspects, there are at least three mutations to the domains that interactwith the PAM region or surrounding sequences on the blocked nucleic acidmolecules selected from mutations to amino acid residues K538, Y542 andK595 in relation to SEQ ID NO:1 and equivalent amino acid residues inorthologs. In some aspects, the variant nucleic acid-guided nucleasecomprises at least one mutation to the domains that interact with thePAM region or surrounding sequences on the blocked nucleic acidmolecules, wherein the at least one mutation is selected from mutationsto amino acid residues K548, N552 and K607 in relation to SEQ ID NO:2;mutations to amino acid residues K534, Y538 and R591 in relation to SEQID NO:3; mutations to amino acid residues K541, N545 and K601 inrelation to SEQ ID NO:4; mutations to amino acid residues K579, N583 andK635 in relation to SEQ ID NO:5; mutations to amino acid residues K613,N617 and K671 in relation to SEQ ID NO:6; mutations to amino acidresidues K613, N617 and K671 in relation to SEQ ID NO:7; mutations toamino acid residues K617, N621 and K678 in relation to SEQ ID NO:8;mutations to amino acid residues K541, N545 and K601 in relation to SEQID NO:9; mutations to amino acid residues K569, N573 and K625 inrelation to SEQ ID NO:10; mutations to amino acid residues K562, N566and K619 in relation to SEQ ID NO:11; mutations to amino acid residuesK645, N649 and K732 in relation to SEQ ID NO:12; mutations to amino acidresidues K548, N552 and K607 in relation to SEQ ID NO:13; mutations toamino acid residues K592, N596 and K653 in relation to SEQ ID NO:14; ormutations to amino acid residues K521, N525 and K577 in relation to SEQID NO:15.

In some aspects, the variant nucleic acid-guided nuclease comprises atleast one mutation to the domains that interact with the PAM region orsurrounding sequences on the blocked nucleic acid molecules, whereinsingle stranded DNA is cleaved 1.2 to 2.5 times faster than doublestranded DNA is cleaved, at least three to four times faster than doublestranded DNA is cleaved, and in some aspects, single stranded DNA iscleaved at least five times faster than double stranded DNA is cleaved.In aspects, the variant nucleic acid-guided nuclease exhibits cis- andtrans-cleavage activity.

In some aspects, the variant nucleic acid-guided nuclease comprises atleast two mutations to the domains that interact with the PAM region orsurrounding sequences on the blocked nucleic acid molecules, and in someaspects, the variant nuclease comprises at least three mutations to thedomains that interact with the PAM region or surrounding sequences onthe blocked nucleic acid molecules.

In any of the embodiments comprising a concentration ratio where blockednucleic acid molecules are at a higher molar concentration than theRNP2s in the reaction mixture, certain aspects provide that theconcentration of the blocked nucleic acid molecules and the RNP2s are ata concentration ratio of at least 1.5 blocked nucleic acid molecules to1 RNP2 in the reaction mixture, and in some aspects, the concentrationof the blocked nucleic acid molecules and the RNP2s are at aconcentration ratio of at least 2 blocked nucleic acid molecules to 1RNP2 in the reaction mixture or at least 3 blocked nucleic acidmolecules to 1 RNP2, or at least 3.5 blocked nucleic acid molecules to 1RNP2, or at least 4 blocked nucleic acid molecules to 1 RNP2, or atleast 4.5 blocked nucleic acid molecules to 1 RNP2, or at least 5blocked nucleic acid molecules to 1 RNP2, or at least 5.5 blockednucleic acid molecules to 1 RNP2, or at least 6 blocked nucleic acidmolecules to 1 RNP2, or at least 6.5 blocked nucleic acid molecules to 1RNP2, or at least 7.5 blocked nucleic acid molecules to 1 RNP2, or atleast 7.5 blocked nucleic acid molecules to 1 RNP2, or at least 8blocked nucleic acid molecules to 1 RNP2, or at least 8.5 blockednucleic acid molecules to 1 RNP2, or at least 9 blocked nucleic acidmolecules to 1 RNP2, or at least 9.5 blocked nucleic acid molecules to 1RNP2, or at least 10 blocked nucleic acid molecules to 1 RNP2.

In further embodiments there is provided a variant Cas12a nucleaseengineered such that single stranded DNA is cleaved faster than doublestranded DNA is cleaved, wherein the variant Cas12a nuclease comprisesat least one mutation to the domains that interact with the PAM regionor surrounding sequences on the blocked nucleic acid molecules andwherein the variant Cas12a nuclease exhibits both cis- andtrans-cleavage activity. In some aspects, wherein the at least onemutation to the domains that interact with the PAM region or surroundingsequences on the blocked nucleic acid molecules is selected frommutations to amino acid residues K538, Y542 and K595 in relation to SEQID NO:1; the at least one mutation to the domains that interact with thePAM region or surrounding sequences on the blocked nucleic acidmolecules is selected from mutations to amino acid residues K548, N552and K607 in relation to SEQ ID NO:2; the at least one mutation to thedomains that interact with the PAM region or surrounding sequences onthe blocked nucleic acid molecules is selected from mutations to aminoacid residues K534, Y538 and R591 in relation to SEQ ID NO:3; the atleast one mutation to the domains that interact with the PAM region orsurrounding sequences on the blocked nucleic acid molecules is selectedfrom mutations to amino acid residues K541, N545 and K601 in relation toSEQ ID NO:4; the at least one mutation to the domains that interact withthe PAM region or surrounding sequences on the blocked nucleic acidmolecules is selected from mutations to amino acid residues K579, N583and K635 in relation to SEQ ID NO:5; the at least one mutation to thedomains that interact with the PAM region or surrounding sequences onthe blocked nucleic acid molecules is selected from mutations to aminoacid residues K613, N617 and K671 in relation to SEQ ID NO:6; the atleast one mutation to the domains that interact with the PAM region orsurrounding sequences on the blocked nucleic acid molecules is selectedfrom mutations to amino acid residues K613, N617 and K671 in relation toSEQ ID NO:7; the at least one mutation to the domains that interact withthe PAM region or surrounding sequences on the blocked nucleic acidmolecules is selected from mutations to amino acid residues K617, N621and K678 in relation to SEQ ID NO:8; the at least one mutation to thedomains that interact with the PAM region or surrounding sequences onthe blocked nucleic acid molecules is selected from mutations to aminoacid residues K541, N545 and K601 in relation to SEQ ID NO:9; the atleast one mutation to the domains that interact with the PAM region orsurrounding sequences on the blocked nucleic acid molecules is selectedfrom mutations to amino acid residues K569, N573 and K625 in relation toSEQ ID NO:10; the at least one mutation to the domains that interactwith the PAM region or surrounding sequences on the blocked nucleic acidmolecules is selected from mutations to amino acid residues K562, N566and K619 in relation to SEQ ID NO:11; the at least one mutation to thedomains that interact with the PAM region or surrounding sequences onthe blocked nucleic acid molecules is selected from mutations to aminoacid residues K645, N649 and K732 in relation to SEQ ID NO:12; the atleast one mutation to the domains that interact with the PAM region orsurrounding sequences on the blocked nucleic acid molecules is selectedfrom mutations to amino acid residues K548, N552 and K607 in relation toSEQ ID NO:13; the at least one mutation to the domains that interactwith the PAM region or surrounding sequences on the blocked nucleic acidmolecules is selected from mutations to amino acid residues K592, N596and K653 in relation to SEQ ID NO:14; or the at least one mutation tothe domains that interact with the PAM region or surrounding sequenceson the blocked nucleic acid molecules is selected from mutations toamino acid residues K521, N525 and K577 in relation to SEQ ID NO:15including and equivalent amino acid residues in Cas12a orthologs tothese SEQ ID Nos: 1-15.

In some aspects, the variant Cas12a nuclease that has been engineeredsuch that single stranded DNA is cleaved faster than double stranded DNAis cleaved comprises any one of SEQ ID NOs: 16-600.

Alternatively, an embodiment provides a single-strand-specific Cas12anucleic acid-guided nucleases comprising an LbCas12a (i.e., SEQ IDNO: 1) with an acetylated K595 (K595K^(Ac)) residue; an AsCas12a (i.e.,SEQ ID NO: 2) with an acetylated K607 (K607K^(Ac)) residue; a CtCas12a(i.e., SEQ ID NO: 3) with an acetylated R591 (R591R A c) residue; anEeCas12a (i.e., SEQ ID NO: 4) with an acetylated K601 (K607K^(Ac))residues; an Mb3Cas12a (i.e., SEQ ID NO: 5) with an acetylated K635(K635K^(Ac)) residue; an FnCas12a (i.e., SEQ ID NO: 6) with anacetylated K671 (K671K^(Ac)) residue; an FnoCas12a (i.e., SEQ ID NO: 7)with an acetylated N671 (N671K^(Ac)) residue; an FbCas12a (i.e., SEQ IDNO: 8) with an acetylated K678 (K678K^(Ac)) residue; an Lb4Cas12a (i.e.,SEQ ID NO: 9) with an acetylated K601 (K601K^(Ac)) residue; an MbCas12a(i.e., SEQ ID NO: 10) with an acetylated K625 (K625K^(Ac)) residue; aPb2Cas12a (i.e., SEQ ID NO: 11) with an acetylated K619 (K619K^(Ac))residue; a PgCas12a (i.e., SEQ ID NO: 12) with an acetylated K732(K732K^(Ac)) residue; an AaCas12a (i.e., SEQ ID NO: 13) with anacetylated K607 (K607K^(Ac)) residue; a BoCas12a (i.e., SEQ ID NO: 14)with an acetylated K653 (K653K^(Ac)) residue; or an CmaCas12a (i.e., SEQID NO: 15) with an acetylated K577 (K577K^(Ac)) residue. Thesingle-strand-specific Cas12a nucleic acid-guided nucleases of thedisclosure may be a Cas12a ortholog acetylated at the amino acid of theortholog equivalent to K595 of SEQ ID NO:1.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1A is an overview of a prior art quantitative PCR (“qPCR”) assaywhere target nucleic acids of interest from a sample are amplifiedbefore detection.

FIG. 1B is an overview of the general principles underlying the nucleicacid-guided nuclease cascade assay described in detail herein wheretarget nucleic acids of interest from a sample do not need to beamplified before detection.

FIG. 1C is an illustration of the unwinding issue that is mitigated bythe modalities described herein.

FIG. 2A is a diagram showing the sequence of steps in an exemplarycascade assay utilizing blocked nucleic acid molecules.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid moleculeand a method for unblocking the blocked nucleic acid molecules of thedisclosure.

FIG. 2C shows schematics of several exemplary blocked nucleic acidmolecules containing the structure of Formula I, as described herein.

FIG. 2D shows schematics of several exemplary blocked nucleic acidmolecules containing the structure of Formula II, as described herein.

FIG. 2E shows schematics of several exemplary blocked nucleic acidmolecules containing the structure of Formula III, as described herein.

FIG. 2F shows schematics of several exemplary blocked nucleic acidmolecules containing the structure of Formula IV, as described herein.

FIG. 2G shows an exemplary single-stranded blocked nucleic acid moleculewith a design able to block R-loop formation with an RNP complex,thereby blocking activation of the trans-nuclease activity of an RNPcomplex (i.e., RNP2).

FIG. 2H shows schematics of exemplary circularized blocked nucleic acidmolecules.

FIG. 3A is a diagram showing the sequence of steps in an exemplarycascade assay involving circular blocked primer molecules and lineartemplate molecules.

FIG. 3B is a diagram showing the sequence of steps in an exemplarycascade assay involving circular blocked primer molecules and circulartemplate molecules.

FIG. 4 illustrates three embodiments of reporter moieties.

FIG. 5 is a simplified block diagram of an exemplary method fordesigning, synthesizing and screening variant nucleic acid-guidednucleases.

FIG. 6A shows the result of protein structure prediction using Rosettaand SWISS modeling of wildtype LbCas12a (Lachnospriaceae bacteriumCas12a).

FIG. 6B shows the result of example mutations on the LbCas12a proteinstructure prediction using Rosetta and SWISS modeling of LbCas12a andindicating the PAM regions.

FIG. 7 is a simplified diagram of acetylating the K595 amino acid in thewildtype sequence of LbCas12a (K595K Ac).

FIG. 8A is an illustration of a blocked nucleic acid molecule with bulkymodifications, cleavage thereof, and steric hindrance at thePAM-interacting (PI) domain in a nucleic acid-guided nuclease caused by5′ and 3′ modifications to a blocked nucleic acid molecule.

FIG. 8B illustrates five exemplary variations of blocked nucleic acidmolecules with bulky modifications.

FIGS. 8C, 8D and 8E list exemplary bulky modifications for 5′, 3′, andinternal positions in blocked nucleic acid molecules.

FIG. 9 is an illustration of a lateral flow assay that can be used todetect the cleavage and separation of a signal from a reporter moiety.

FIG. 10A depicts Molecule U29 and describes the properties thereof,where MU29 was used to generate the data shown in FIGS. 10B-10H.

FIG. 11A shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutation G532A in thewildtype sequence.

FIG. 11B shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutation K538A in thewildtype sequence.

FIG. 11C shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutation Y542A in thewildtype sequence.

FIG. 11D shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutation K595A in thewildtype sequence.

FIG. 11E shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutations G532A, K538A,Y5442A and K595A in the wildtype sequence.

FIG. 11F shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutation K595D in thewildtype sequence.

FIG. 11G shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutation K595E in thewildtype sequence.

FIG. 11H shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutations K538A, Y542A andK595D in the wildtype sequence.

FIG. 11I shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutations K538A, Y542A andK595E in the wildtype sequence.

FIGS. 12A-12G are a series of graphs showing the time for detection ofdsDNA and ssDNA both with and without PAM sequences for wildtypeLbaCas12a and engineered variants of LbaCas12a.

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

Definitions

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention. The terms used herein are intended to have the plain andordinary meaning as understood by those of ordinary skill in the art.

All of the functionalities described in connection with one embodimentof the compositions and/or methods described herein are intended to beapplicable to the additional embodiments of the compositions and/ormethods except where expressly stated or where the feature or functionis incompatible with the additional embodiments. For example, where agiven feature or function is expressly described in connection with oneembodiment but not expressly mentioned in connection with an alternativeembodiment, it should be understood that the feature or function may bedeployed, utilized, or implemented in connection with the alternativeembodiment unless the feature or function is incompatible with thealternative embodiment.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “a system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth. Additionally, it is to be understood that terms such as“left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”“outer” that may be used herein merely describe points of reference anddo not necessarily limit embodiments of the present disclosure to anyparticular orientation or configuration. Furthermore, terms such as“first,” “second,” “third,” etc., merely identify one of a number ofportions, components, steps, operations, functions, and/or points ofreference as disclosed herein, and likewise do not necessarily limitembodiments of the present disclosure to any particular configuration ororientation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention. Conventional methodsare used for the procedures described herein, such as those provided inthe art, and demonstrated in the Examples and various generalreferences. Unless otherwise stated, nucleic acid sequences describedherein are given, when read from left to right, in the 5′ to 3′direction. Nucleic acid sequences may be provided as DNA, as RNA, or acombination of DNA and RNA (e.g., a chimeric nucleic acid).

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in smaller ranges, and arealso encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both limits, ranges excluding either or both of those included limitsare also included in the invention.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the multiple specified features or components withor without another. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the term “about,” as applied to one or more values ofinterest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1%, or less in either direction (greater than or less than)of a stated reference value, unless otherwise stated or otherwiseevident from the context (except where such number would exceed 100% ofa possible value).

As used herein, the terms “binding affinity” or “dissociation constant”or “K_(d)” refer to the tendency of a molecule to bind (covalently ornon-covalently) to a different molecule. A high K_(d) (which in thecontext of the present disclosure refers to blocked nucleic acidmolecules or blocked primer molecules binding to RNP2) indicates thepresence of more unbound molecules, and a low K_(d) (which in thecontext of the present disclosure refers to unblocked nucleic acidmolecules or unblocked primer molecules binding to RNP2) indicates thepresence of more bound molecules. In the context of the presentdisclosure and the binding of blocked or unblocked nucleic acidmolecules or blocked or unblocked primer molecules to RNP2, low K_(d)values are in a range from about 100 fM to about 1 aM or lower (e.g.,100 zM) and high K_(d) values are in the range of 100 nM-100 μM (10 mM)and thus are about 10⁵- to 10¹⁰-fold or higher as compared to low K_(d)values.

As used herein, the terms “binding domain” or “binding site” refer to aregion on a protein, DNA, or RNA, to which specific molecules and/orions (ligands) may form a covalent or non-covalent bond. By way ofexample, a polynucleotide sequence present on a nucleic acid molecule(e.g., a primer binding domain) may serve as a binding domain for adifferent nucleic acid molecule (e.g., an unblocked primer nucleic acidmolecule). Characteristics of binding sites are chemical specificity, ameasure of the types of ligands that will bond, and affinity, which is ameasure of the strength of the chemical bond.

As used herein, the term “blocked nucleic acid molecule” refers tonucleic acid molecules that cannot bind to the first or second RNPcomplex to activate cis- or trans-cleavage. “Unblocked nucleic acidmolecule” refers to a formerly blocked nucleic acid molecule that canbind to the second RNP complex (RNP2) to activate trans-cleavage ofadditional blocked nucleic acid molecules. A “blocked nucleic acidmolecule” may be a “blocked primer molecule” in some embodiments of thecascade assay.

The terms “Cas RNA-guided nucleic acid-guided nuclease” or “CRISPRnuclease” or “nucleic acid-guided nuclease” refer to a CRISPR-associatedprotein that is an RNA-guided nucleic acid-guided nuclease suitable forassembly with a sequence-specific gRNA to form a ribonucleoprotein (RNP)complex.

As used herein, the terms “cis-cleavage”, “cis-nucleic acid-guidednuclease activity”, “cis-mediated nucleic acid-guided nucleaseactivity”, “cis-nuclease activity”, “cis-mediated nuclease activity”,and variations thereof refer to sequence-specific cleavage of a targetnucleic acid of interest, including an unblocked nucleic acid moleculeor synthesized activating molecule, by a nucleic acid-guided nuclease inan RNP complex. Cis-cleavage is a single turn-over cleavage event inthat only one substrate molecule is cleaved per event.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen-bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” or“percent homology” to a specified second nucleotide sequence. Forexample, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10, or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-ATCGAT-5′is 100% complementary to a region of the nucleotide sequence5′-GCTAGCTAG-3′.

As used herein, the term “contacting” refers to placement of twomoieties in direct physical association, including in solid or liquidform. Contacting can occur in vitro with isolated cells (for example ina tissue culture dish or other vessel) or in samples or in vivo byadministering an agent to a subject.

The term “conservative amino acid substitution” refers to theinterchangeability in proteins of amino acid residues having similarside chains. For example, a group of amino acids having aliphatic sidechains comprises glycine, alanine, valine, leucine, and isoleucine; agroup of amino acids having aliphatic-hydroxyl side chains comprisesserine and threonine; a group of amino acids having amide containingside chains comprises asparagine and glutamine; a group of amino acidshaving aromatic side chains comprises phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains compriseslysine, arginine, and histidine; a group of amino acids having acidicside chains comprises glutamate and aspartate; and a group of aminoacids having sulfur containing side chains comprises cysteine andmethionine. Exemplary conservative amino acid substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine-glycine, and asparagine-glutamine.

A “control” is a reference standard of a known value or range of values.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a crRNA region or guide sequence capable ofhybridizing to the target strand of a target nucleic acid of interest,and 2) a scaffold sequence capable of interacting or complexing with anucleic acid-guided nuclease. The crRNA region of the gRNA is acustomizable component that enables specificity in every nucleicacid-guided nuclease reaction. A gRNA can include any polynucleotidesequence having sufficient complementarity with a target nucleic acid ofinterest to hybridize with the target nucleic acid of interest and todirect sequence-specific binding of a ribonucleoprotein (RNP) complexcontaining the gRNA and nucleic acid-guided nuclease to the targetnucleic acid. Target nucleic acids of interest may include a protospaceradjacent motif (PAM), and, following gRNA binding, the nucleicacid-guided nuclease induces a double-stranded break either inside oroutside the protospacer region on the target nucleic acid of interest,including on an unblocked nucleic acid molecule or synthesizedactivating molecule. A gRNA may contain a spacer sequence including aplurality of bases complementary to a protospacer sequence in the targetnucleic acid. For example, a spacer can contain about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, ormore bases. The gRNA spacer may be 50%, 60%, 75%, 80%, 85%, 90%, 95%,97.5%, 98%, 99%, or more complementary to its corresponding targetnucleic acid of interest. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences. A guide RNA may befrom about 20 nucleotides to about 300 nucleotides long. Guide RNAs maybe produced synthetically or generated from a DNA template.

“Modified” refers to a changed state or structure of a molecule.Molecules may be modified in many ways including chemically,structurally, and functionally. In one embodiment, a nucleic acidmolecule (for example, a blocked nucleic acid molecule) may be modifiedby the introduction of non-natural nucleosides, nucleotides, and/orinternucleoside linkages. In another embodiment, a modified protein(e.g., a modified or variant nucleic acid-guided nuclease) may refer toany polypeptide sequence alteration which is different from thewildtype.

The terms “percent sequence identity”, “percent identity”, or “sequenceidentity” refer to percent (%) sequence identity with respect to areference polynucleotide or polypeptide sequence following alignment bystandard techniques. Alignment for purposes of determining percentsequence identity can be achieved in various ways that are within thecapabilities of one of skill in the art, for example, using publiclyavailable computer software such as BLAST, BLAST-2, PSI-BLAST, orMegalign software. In some embodiments, the software is MUSCLE (Edgar,Nucleic Acids Res., 32(5):1792-1797 (2004)). Those skilled in the artcan determine appropriate parameters for aligning sequences, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For example, in embodiments, percentsequence identity values are generated using the sequence comparisoncomputer program BLAST (Altschul, et al., J. Mol. Biol., 215:403-410(1990)).

As used herein, the terms “preassembled ribonucleoprotein complex”,“ribonucleoprotein complex”, “RNP complex”, or “RNP” refer to a complexcontaining a guide RNA (gRNA) and a nucleic acid-guided nuclease, wherethe gRNA is integrated with the nucleic acid-guided nuclease. The gRNA,which includes a sequence complementary to a target nucleic acid ofinterest, guides the RNP to the target nucleic acid of interest andhybridizes to it. The hybridized target nucleic acid-gRNA units arecleaved by the nucleic acid-guided nuclease. In the cascade assaysdescribed herein, a first ribonucleoprotein complex (RNP1) includes afirst guide RNA (gRNA) specific to a target nucleic acid of interest,and a first nucleic acid-guided nuclease, such as, for example, cas12aor cas14a for a DNA target nucleic acid, or cas13a for an RNA targetnucleic acid. A second ribonucleoprotein complex (RNP2) for signalamplification includes a second guide RNA specific to an unblockednucleic acid or synthesized activating molecule, and a second nucleicacid-guided nuclease, which may be different from or the same as thefirst nucleic acid-guided nuclease.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably. Proteins may or may not be made up entirely of aminoacids.

As used herein, the term “sample” refers to tissues; cells or componentparts; body fluids, including but not limited to peripheral blood,serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum,saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid,cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostaticfluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter,hair, tears, cyst fluid, pleural and peritoneal fluid, pericardialfluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus,sebum, vomit, vaginal secretions, mucosal secretion, stool water,pancreatic juice, lavage fluids from sinus cavities, bronchopulmonaryaspirates, blastocyl cavity fluid, and umbilical cord blood. “Sample”may also refer to specimens or aliquots from food; agriculturalproducts; pharmaceuticals; cosmetics, nutraceuticals; personal careproducts; environmental substances such as soil, water (from bothnatural and treatment sites), air, or sewer samples; industrial sitesand products; and chemicals and compounds. A sample further may includea homogenate, lysate or extract. A sample further refers to a medium,such as a nutrient broth or gel, which may contain cellular components,such as proteins or nucleic acid molecules.

The terms “target DNA sequence”, “target sequence”, “target nucleic acidof interest”, “target molecule of interest”, “target nucleic acid”, or“target of interest” refer to any locus that is recognized by a gRNAsequence in vitro or in vivo. The “target strand” of a target nucleicacid of interest is the strand of the double-stranded target nucleicacid that is complementary to a gRNA. The spacer sequence of a gRNA maybe 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99% or morecomplementary to the target nucleic acid of interest. Optimal alignmentcan be determined with the use of any suitable algorithm for aligningsequences. Full complementarity is not necessarily required providedthere is sufficient complementarity to cause hybridization andtrans-cleavage activation of an RNP complex. A target nucleic acid ofinterest can include any polynucleotide, such as DNA (ssDNA or dsDNA) orRNA polynucleotides. A target nucleic acid of interest may be located inthe nucleus or cytoplasm of a cell such as, for example, within anorganelle of a eukaryotic cell, such as a mitochondrion or achloroplast, or it can be exogenous to a host cell, such as a eukaryoticcell or a prokaryotic cell. The target nucleic acid of interest may bepresent in a sample, such as a biological or environmental sample, andit can be a viral nucleic acid molecule, a bacterial nucleic acidmolecule, a fungal nucleic acid molecule, or a polynucleotide of anotherorganism, such as a coding or a non-coding sequence, and it may includesingle-stranded or double-stranded DNA molecules, such as a cDNA orgenomic DNA, or RNA molecules, such as mRNA, tRNA, and rRNA. The targetnucleic acid of interest may be associated with a protospacer adjacentmotif (PAM) sequence, which may include a 2-5 base pair sequenceadjacent to the protospacer. In some embodiments 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more target nucleic acids can be detected by the disclosedmethod.

As used herein, the terms “trans-cleavage”, “trans-nucleic acid-guidednuclease activity”, “trans-mediated nucleic acid-guided nucleaseactivity”, “trans-nuclease activity”, “trans-mediated nuclease activity”and variations thereof refer to indiscriminate, non-sequence-specificcleavage of a target nucleic acid molecule by a nucleic acid-guidednuclease (such as by a Cas12, Cas13, and Cas14) which is triggered bybinding of N nucleotides of a target nucleic acid molecule to a gRNAand/or by cis-(sequence-specific) cleavage of a target nucleic acidmolecule. Trans-cleavage is a “multiple turn-over” event, in that morethan one substrate molecule is cleaved after initiation by a singleturn-over cis-cleavage event.

Type V CRISPR/Cas nucleic acid-guided nucleases are a subtype of Class 2CRISPR/Cas effector nucleases such as, but not limited to, engineeredCas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e),CasY (Cas12d), Cas 13a nucleases or naturally-occurring proteins, suchas a Cas12a isolated from, for example, Francisella tularensis subsp.novicida (Gene ID: 60806594), Candidatus Methanoplasma termitum (GeneID: 24818655), Candidatus Methanomethylophilus alvus (Gene ID:15139718), and [Eubacterium] eligens ATCC 27750 (Gene ID: 41356122), andan artificial polypeptide, such as a chimeric protein.

The term “variant” in the context of the present disclosure refers to apolypeptide or polynucleotide that differs from a reference polypeptideor polynucleotide but retains essential properties. A typical variant ofa polypeptide differs in amino acid sequence from another referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many if not most regions, identical. A variant and referencepolypeptide may differ in one or more amino acid residues (e.g.,substitutions, additions, and/or deletions). A variant of a polypeptidemay be a conservatively modified variant. A substituted or insertedamino acid residue may or may not be one encoded by the genetic code(e.g., a non-natural amino acid). A variant of a polypeptide may benaturally occurring, such as an allelic variant, or it may be a variantthat is not known to occur naturally. Variants includemodifications—including chemical modifications—to one or more aminoacids that do not involve amino acid substitutions, additions ordeletions.

As used herein, the terms “variant engineered nucleic acid-guidednuclease” or “variant nucleic acid-guided nuclease” refer to nucleicacid-guided nucleases have been engineered to mutate the PAM interactingdomains in the LbCas12a (Lachnospriaceae bacterium Cas12a), AsCas 12a(Acidaminococcus sp. BV3L6 Cas12a), CtCas12a (Candidatus Methanoplasmatermitum Cas12a), EeCas 12a (Eubacterium eligens Cas12a), Mb3Cas12a(Moraxella bovoculi Cas12a), FnCas12a (Francisella novicida Cas12a),FnoCas12a (Francisella tularensis subsp. novicida FTG Cas12a), FbCas12a(Flavobacteriales bacterium Cas12a), Lb4Cas12a (Lachnospira eligensCas12a), MbCas12a (Moraxella bovoculi Cas12a), Pb2Cas12a (Prevotellabryantii Cas12a), PgCas12a (Candidatus Parcubacteria bacterium Cas12a),AaCas12a (Acidaminococcus sp. Cas12a), BoCas12a (Bacteroidetes bacteriumCas12a), CMaCas12a (Candidatus Methanomethylophilus alvus Mx1201Cas12a), and to-be-discovered equivalent Cas12a nucleic acid-guidednucleases such that double-stranded DNA (dsDNA) substrates bind to thevariant nucleic acid-guided nuclease and are cleaved by the variantnucleic acid-guided nuclease at a slower rate than single-stranded DNA(ssDNA) substrates.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, synthetic chromosomes, and the like.

DETAILED DESCRIPTION

The present disclosure provides compositions of matter and methods forcascade assays that detect nucleic acids. The cascade assays allow formassive multiplexing, and provide high accuracy, low cost, minimumworkflow and results in less than one minute or, in some embodiments,virtually instantaneously, even at ambient temperatures of about 16-20°C. or less up to 48° C. The cascade assays described herein comprisefirst and second ribonucleoprotein complexes and either blocked nucleicacid molecules or blocked primer molecules. The blocked nucleic acidmolecules or blocked primer molecules keep the second ribonucleoproteincomplexes “locked” unless and until a target nucleic acid of interestactivates the first ribonucleoprotein complex. The methods comprise thesteps of providing cascade assay components, contacting the cascadeassay components with a sample, and detecting a signal that is generatedonly when a target nucleic acid of interest is present in the sample.

Early and accurate identification of, e.g., infectious agents, microbecontamination, variant nucleic acid sequences that indicate the presenceof diseases such as cancer or contamination by heterologous sources isimportant in order to select correct treatment; identify tainted food,pharmaceuticals, cosmetics and other commercial goods; and to monitorthe environment. Nucleic acid-guided nucleases, such as Type V nucleicacid-guided nucleases, can be utilized for the detection of targetnucleic acids of interest associated with diseases, food contaminationand environmental threats. However, currently available nucleic aciddetection such as quantitative PCR (also known as real time PCR or qPCR)or CRISPR-based detection assays such as SHERLOCK™ and DETECTR™ rely onDNA amplification, which requires time and may lead to changes to therelative proportion of nucleic acids, particularly in multiplexednucleic acid assays. The lack of rapidity for these detection assays isdue to the fact that there is a significant lag phase early in theamplification process where fluorescence above background cannot bedetected. With qPCR, for example, there is a lag until the cyclethreshold or Ct value, which is the number of amplification cyclesrequired for the fluorescent signal to exceed the background level offluorescence, is achieved and can be quantified.

The present disclosure describes a signal boost cascade assay andimprovements thereto that can detect one or more target nucleic acids ofinterest (e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower)limits in less than one minute and in some embodiments virtuallyinstantaneously without the need for amplifying the target nucleicacid(s) of interest, thereby avoiding the drawbacks of multiplexamplification, such as primer-dimerization. As described in detailbelow, the cascade assays utilize signal boost mechanisms comprisingvarious components including nucleic acid-guided nucleases, guide RNAs(gRNAs) incorporated into ribonucleoprotein complexes (RNP complexes),blocked nucleic acid molecules or blocked primer molecules, reportermoieties, and, in some embodiments, polymerases and template molecules.A particularly advantageous feature of the cascade assay is that, withthe exception of the gRNA in RNP1 (i.e., gRNA1), the cascade assaycomponents are essentially identical no matter what target nucleicacid(s) of interest are being detected, and gRNA1 is easilyprogrammable.

The improvements to the signal amplification or signal boost cascadeassay described herein result from preventing undesired unwinding of theblocked nucleic acid molecules in the reaction mix by the secondribonucleoprotein complex (RNP2) before the blocked nucleic acidmolecules are unblocked via trans-cleavage, leading to increasedefficiency, reduced background, and increased signal-to-noise ratio inthe cascade assay. Minimizing undesired unwinding serves two purposes.First, preventing undesired unwinding that happens not as a result ofunblocking due to trans-cleavage subsequent to cis-cleavage of thetarget nucleic acid of interest or trans-cleavage of unblocked nucleicacid molecules—but due to other factors—leads to a “leaky” cascade assaysystem, which in turn leads to non-specific signal generation.

Second, preventing undesired unwinding limits non-specific interactionsbetween the nucleic acid-guided nucleases (here, in the RNP2s) andblocked nucleic acid molecules such that only blocked nucleic acidmolecules that become unblocked due to trans-cleavage activity reactwith the nucleic acid-guided nucleases. This “fidelity” in the cascadeassay leads primarily to desired interactions and limits “wasteful”interactions where the nucleic acid-guided nucleases are essentiallyacting on blocked nucleic acid molecules rather than unblocked nucleicacid molecules. That is, the nucleic acid-guided nucleases are focusedon desired interactions which then leads to immediate signalamplification or boost in the cascade assay.

The present disclosure provides three modalities to minimize leakinessleading to minimal false positives or higher background signal. Thepresent disclosure demonstrates that undesired unwinding of the blockednucleic acid molecules can be lessened substantially by 1) increasingthe molar ratio of the concentration of blocked nucleic acid molecules(equivalent to a target nucleic acid molecule for the RNP2) to be equalto or greater than the molar concentration of RNP2 (e.g., the nucleicacid-guided nuclease in RNP2); 2) engineering the nucleic acid-guidednuclease used in RNP2 so as to increase the time it takes the nucleicacid-guided nuclease to recognize double-strand DNA at least two-foldand preferably three-fold or more; and/or 3) engineering the blockednucleic acid molecules to include bulky modifications (that is,molecules with a size of about 1 nm or less).

The first modality for minimizing undesired unwinding of the blockednucleic acid molecules (or blocked primer molecules) is to adjust therelative concentrations of the blocked nucleic acid molecules (orblocked primer molecules) and RNP2s such that the molar concentration ofthe blocked nucleic acid molecules (or blocked primer molecules) isequal to or greater than the molar concentration of RNP2s. Before thepresent disclosure, the common wisdom in performing CRISPR detectionassays was to use a vast excess of nucleic acid-guided nuclease (e.g.,RNP complex) to target.

In most detection assays, the quantity of the target nucleic acid ofinterest is not known (e.g., the detection assay is performed on asample with an unknown concentration of target); however, in experimentsconducted to determine the level of detection of two CRISPR detectionassays known in the art, DETECTR™ and SHERLOCK™, the nucleic acidnuclease was present at ng/μL concentrations and the target of interestwas present at very low copy numbers or at femtomolar to attamolarconcentration. Thus, the present methods and reagent mixtures not onlyadjust the relative concentrations of the blocked nucleic acid molecules(or blocked primer molecules) and RNP2s such that the molarconcentration of the blocked nucleic acid molecules (or blocked primermolecules) is equal to or greater than the molar concentration of RNP2s,but the molar concentration of RNP2s may still exceed the molarconcentration of the blocked nucleic acid molecules by a lesser amount,such as where the molar concentration of RNP2s exceeds the molarconcentration of blocked nucleic acid molecules (or blocked targetmolecules) by 100,000×, 50,000×, 25,000×, 10,000×, 5,000×, 1000×, 500×,100×, or 10× or less.

For example, Sun, et al. ran side-by-side comparisons of the DETECTR™and SHERLOCK™ detection assays, using a concentration of 100 ng/μLLbCas12a in the DETECTR™ assay and a concentration of 20 ng/μL LwCas13ain the SHERLOCK™ assay, where the concentration of the target nucleicacid molecules ranged from 0 copies/μL, 0.1 copies/μL, 0.2 copies/μL,1.0 copy/μL, 2.0 copies/μL, 5.0 copies/μL, 10.0 copies/μL, and so on upto 200.0 copies/μL. (Sun, et al., J. of Translational Medicine, 12:74(2021).) In addition, Broughton, et al., ran the DETECTR™ assay using aconcentration range of 2.5 copies/pi, to 1250 copies/μL target nucleicacid molecules to nM LbCas12 (see, Broughton, et al., Nat. Biotech.,38:870-74 (2020)); and Lee, et al., ran the SHERLOCK™ assay using aconcentration range of 10 fM to 50 aM target nucleic acid molecules to150 nM Cas12 (see Lee, et al., PNAS, 117(41):25722-31 (2020). Thus, theratio of nucleic acid-guided nuclease to blocked nucleic acid molecule(e.g., target for RNP2) described herein is very different from ratiospracticed in the art and this ratio has been determined to limitundesired unwinding of the blocked nucleic acid molecules (or blockedprimer molecules).

In a second modality, variant nucleic acid-guided nucleases have beenengineered to mutate the domains in the variants that interact with thePAM region or surrounding sequences on the blocked nucleic acidmolecules in, e.g., Type V nucleic acid-guided nucleases such as theLbCas12a (Lachnospriaceae bacterium Cas12a), AsCas 12a (Acidaminococcussp. BV3L6 Cas12a), CtCas12a (Candidatus Methanoplasma termitum Cas12a),EeCas12a (Eubacterium eligens Cas12a), Mb3Cas12a (Moraxella bovoculiCas12a), FnCas12a (Francisella novicida Cas12a), FnoCas12a (Francisellatularensis subsp. novicida FTG Cas12a), FbCas12a (Flavobacterialesbacterium Cas12a), Lb4Cas12a (Lachnospira eligens Cas12a), MbCas12a(Moraxella bovoculi Cas12a), Pb2Cas12a (Prevotella bryantii Cas12a),PgCas12a (Candidatus Parcubacteria bacterium Cas12a), AaCas12a(Acidaminococcus sp. Cas12a), BoCas12a (Bacteroidetes bacterium Cas12a),CMaCas12a (Candidatus Methanomethylophilus alvus Mx1201 Cas12a), andother related nucleic acid-guided nucleases (e.g., homologs andorthologs of these nucleic acid-guided nucleases) also limit unwinding.These variant nucleic acid-guided nucleases have been engineered suchthat double-stranded DNA (dsDNA) substrates bind to and activate to thevariant nucleic acid-guided nucleases slowly, but single-stranded DNA(ssDNA) substrates continue to bind and activate the variant nucleicacid-guided nuclease at a high rate. Thus, the variant nucleicacid-guided nucleases effect a “lock” on the RNP complex (here, theRNP2) vis-à-vis double-strand DNA. Locking RNP2 in this way lessens thelikelihood of undesired unwinding of the blocked nucleic acid moleculesas described in detail herein (see FIG. 1C and the accompanyingdiscussion). Modifying the nucleic acid-guided nucleases to notrecognize dsDNA or to recognize dsDNA is contrary to what is desired inother CRISPR-based diagnostic/detection assays.

Finally, another modality for minimizing undesired unwinding of theblocked nucleic acid molecules is to use “bulky modifications” at the 5′and/or 3′ ends of the blocked nucleic acid molecules and/or at internalnucleic acid bases of the blocked nucleic acid molecules. Doing socreates steric hindrance at the domains of the nucleic acid-guidednuclease in RNP2 that interact with the PAM region or that interact withsurrounding sequences on the blocked nucleic acid molecules, disrupting,e.g., PAM recognition in the target strand and preventing displacementof the non-target strand. Using bulky modifications is yet another pathto locking RNP2 to double-strand DNA molecules thereby lessening thelikelihood of undesired unwinding of the blocked nucleic acid moleculesas described in detail herein (again, see FIG. 1C and the accompanyingdiscussion). “Bulky modifications” include molecules with a size ofabout 1 nm or less.

FIG. 1A provides a simplified diagram demonstrating a prior art methodfor quantifying target nucleic acids of interest in a sample; namely,the quantitative polymerase chain reaction or qPCR, which to date may beconsidered the gold standard for quantitative detection assays. Thedifference between PCR and qPCR is that PCR is a qualitative techniquethat indicates the presence or absence of a target nucleic acid ofinterest in a sample, where qPCR allows for quantification of targetnucleic acids of interest in a sample. qPCR involves selectiveamplification and quantitative detection of specific regions of DNA orcDNA (i.e., the target nucleic acid of interest) using oligonucleotideprimers that flank the specific region(s) in the target nucleic acid(s)of interest. The primers are used to amplify the specific regions usinga polymerase. Like PCR, repeated cycling of the amplification processleads to an exponential increase in the number of copies of theregion(s) of interest; however, unlike traditional PCR, the increase istracked using an intercalating dye or, as shown in FIG. 1A, asequence-specific probe (e.g., a “Taq-man probe”) the fluorescence ofwhich is detected in real time. RT-qPCR differs from qPCR in that areverse transcriptase is used to first copy RNA molecules to producecDNA before the qPCR process commences.

FIG. 1A is an overview of a qPCR assay where target nucleic acids ofinterest from a sample are amplified before detection. FIG. 1A shows theqPCR method 10, comprising a double-stranded DNA template 12 and asequence specific Taq-man probe 14 comprising a region complementary tothe target nucleic acid of interest 20, a quencher 16, a quenchedfluorophore 18 where 22 denotes quenching between the quencher 16 andquenched fluorophore 18. Upon denaturation, the two strands of thedouble-stranded DNA template 12 separate into complementary singlestrands 26 and 28. In the next step, primers 24 and 24′ anneal tocomplementary single strands 26 and 28, as does the sequence-specificTaq-man probe 14 via the region complementary 20 to the complementarystrand 26 of the target nucleic acid of interest. Initially the Taq-manprobe is annealed to complementary strand 26 of the target region ofinterest intact; however, primers 24 and 24′ are extended by polymerase30 but the Taq-man probe is not, due to the absence of a 3′ hydroxygroup. Instead, the exonuclease activity of the polymerase “chews up”the Taq-man probe, thereby separating the quencher 16 from the quenchedfluorophore 18 resulting in an unquenched or excited-state fluorophore34. The fluorescence quenching ensures that fluorescence occurs onlywhen target nucleic acids of interest are present and being copied,where the fluorescent signal is proportional to the number ofsingle-strand target nucleic acids being amplified.

As noted above, the downside to the prior art, currently availabledetection assays such as qPCR, as well as CRISPR-based reaction assayssuch as SHERLOCK™ and DETECTR™ is that these assays rely on DNAamplification, which, in addition to issues with multiplexing,significantly hinders the ability to perform rapid testing, e.g., in thefield. That is, where the present cascade assay works at ambienttemperatures, including room temperatures and below, assays that requireamplification of the target nucleic acids of interest do not work wellat lower temperatures—even those assays utilizing isothermalamplification—due to non-specific binding of the primers and lowpolymerase activity. Further, primer design is far more challenging. Asfor the lack of rapidity of detection assays that require amplificationof the target nucleic acids of interest, a significant lag phase occursearly in the amplification process where fluorescence above backgroundcannot be detected, particularly in samples with very low copy numbersof the target nucleic acid of interest. And, again, amplification,particularly multiplex amplification, may cause changes to the relativeproportion of nucleic acids in samples that, in turn, lead to artifactsor inaccurate results.

FIG. 1B provides a simplified diagram demonstrating a method (100) of acascade assay. The cascade assay is initiated when the target nucleicacid of interest (104) binds to and activates a first pre-assembledribonucleoprotein complex (RNP1) (102). A ribonucleoprotein complexcomprises a guide RNA (gRNA) and a nucleic acid-guided nuclease, wherethe gRNA is integrated with the nucleic acid-guided nuclease. The gRNA,which includes a sequence complementary to the target nucleic acid ofinterest, guides an RNP complex to the target nucleic acid of interestand hybridizes to it. Typically, preassembled RNP complexes are employedin the reaction mix—as opposed to separate nucleic acid-guided nucleasesand gRNAs—to facilitate rapid (and in the present cascade assays,virtually instantaneous) detection of the target nucleic acid(s) ofinterest.

“Activation” of RNP1 refers to activating trans-cleavage activity of thenucleic acid-guided nuclease in RNP1 (106) by binding of the targetnucleic acid-guided nuclease to the gRNA of RNP1, initiatingcis-cleavage where the target nucleic acid of interest is cleaved by thenucleic acid-guided nuclease. This binding and/or cis-cleavage activitythen initiates trans-cleavage activity (i.e., multi-turnover activity)of the nucleic acid-guided nuclease, where trans-cleavage isindiscriminate, leading to non-sequence-specific cutting of nucleic acidmolecules by the nucleic acid-guided nuclease of RNP1 (102). Thistrans-cleavage activity triggers activation of blocked ribonucleoproteincomplexes (RNP2s) (108) in various ways, which are described in detailbelow. Each newly activated RNP2 (110) activates more RNP2 (108→110),which in turn cleave reporter moieties (112). The reporter moieties(112) may be a synthetic molecule linked or conjugated to a quencher(114) and a fluorophore (116) such as, for example, a probe with a dyelabel (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end.The quencher (114) and fluorophore (116) can be about 20-30 bases apart(or about 10-11 nm apart) or less for effective quenching viafluorescence resonance energy transfer (FRET). Reporter moieties alsoare described in greater detail below.

As more RNP2s are activated (108→110), more trans-cleavage activity isactivated and more reporter moieties are activated (where here,“activated” means unquenched); thus, the binding of the target nucleicacid of interest (104) to RNP1 (102) initiates what becomes a cascade ofsignal production (120), which increases exponentially; hence, the terms“signal amplification” or “signal boost.” The cascade assay thuscomprises a single turnover event that triggers a multi-turnover eventthat then triggers another multi-turnover event in a “cascade.” Asdescribed below in relation to FIG. 4 , the reporter moieties (112) maybe provided as molecules that are separate from the other components ofthe nucleic acid-guided nuclease cascade assay, or the reporter moietiesmay be covalently or non-covalently linked to the blocked nucleic acidmolecules or synthesized activating molecules (i.e., the targetmolecules for the RNP2).

As described in detail below, the present description presents threemodalities for minimizing undesired unwinding of the blocked nucleicacid molecules (or blocked primer molecules), which possess regions ofdouble-strand DNA, where such unwinding can lead to non-specific signalgeneration and false positives. The modalities are 1) altering the ratioof the nucleic acid-guided nuclease in RNP2 to the blocked nucleic acidmolecules in contravention to the common wisdom for CRISPRdetection/diagnostic assays; 2) engineering the nucleic acid-guidednuclease used in RNP2 so that recognition of double-stranded DNA occursmore slowly than for single-strand DNA, in contravention to nucleicacid-guided nucleases that are used in other CRISPR-based detectionassays; and 3) modifying the 5′ and/or 3′ ends and/or various internalnucleic acid bases of the blocked nucleic acid molecules. One, two orall three of these modalities may be employed in a given assay.

FIG. 1C is an illustration of the effects of unwinding. FIG. 1C shows atleft a double-strand blocked nucleic acid molecule comprising a targetstrand and a non-target strand, where the non-target strand comprisesregions (shown as loops) unhybridized to the target strand. Proceedingright at top, cleavage of the loops in the non-target strand bytrans-cleavage initiated by RNP1 or RNP2 destabilizes the double-strandblocked nucleic acid molecule; that is, the now short regions of thenon-target strand that are hybridized to the target strand becomedestabilized and dehybridize. As these short regions dehybridize, thetarget strand is released and can bind to gRNA2 in RNP2, triggeringcis-cleavage of the target strand followed by trans-cleavage ofadditional blocked nucleic acid molecules. This process is the signalboost assay working as designed.

The pathway at the bottom of FIG. 1C illustrates the effect of undesiredunwinding; that is, unwinding due not to trans-cleavage as designed butby other unwinding due to recognition of the blocked nucleic acidmolecule by gRNA2 and the nucleic acid-guided nuclease in RNP2. As seenin the alternative pathway at bottom of FIG. 1C, R-loop formationbetween RNP2 and the blocked nucleic acid molecule (or blocked primermolecule) can still occur due to unwinding of the blocked nucleic acidmolecule after gRNA2 identifies the PAM. Indeed, this unwinding canoccur even in the absence of a PAM. It is an inherent characteristic ofthe biology of nucleic acid-guided nucleases.

Various components of the cascade assay, descriptions of how the cascadeassays work, and the modalities used to minimize undesired unwinding ofthe blocked nucleic acid molecules (or blocked primer molecules) aredescribed in detail below.

Target Nucleic Acids of Interest

The target nucleic acid of interest may be a DNA, RNA, or cDNA molecule.Target nucleic acids of interest may be isolated from a sample ororganism by standard laboratory techniques or may be synthesized bystandard laboratory techniques (e.g., RT-PCR). The target nucleic acidsof interest are identified in a sample, such as a biological sample froma subject (including non-human animals or plants), items of manufacture,or an environmental sample (e.g., water or soil). Non-limiting examplesof biological samples include blood, serum, plasma, saliva, mucus, anasal swab, a buccal swab, a cell, a cell culture, and tissue. Thesource of the sample could be any mammal, such as, but not limited to, ahuman, primate, monkey, cat, dog, mouse, pig, cow, horse, sheep, andbat. Samples may also be obtained from any other source, such as air,water, soil, surfaces, food, beverages, nutraceuticals, clinical sitesor products, industrial sites (including food processing sites) andproducts, plants and grains, cosmetics, personal care products,pharmaceuticals, medical devices, agricultural equipment and sites, andcommercial samples.

In some embodiments, the target nucleic acid of interest is from aninfectious agent (e.g., a bacteria, protozoan, insect, worm, virus, orfungus) that affects mammals, including humans. As a non-limitingexample, the target nucleic acid of interest could be one or morenucleic acid molecules from bacteria, such as Bordetella parapertussis,Bordetella pertussis, Chlamydia pneumoniae, Legionella pneumophila,Mycoplasma pneumoniae, Acinetobacter calcoaceticus-baumannii complex,Bacteroides fragilis, Enterobacter cloacae complex, Escherichia coli,Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae group,Moraxella catarrhalis, Proteus spp., Salmonella enterica, Serratiamarcescens, Haemophilus influenzae, Neisseria meningitidis, Pseudomonasaeruginosa, Stenotrophomonas maltophilia, Enterococcus faecalis,Enterococcus faecium, Listeria monocytogenes, Staphylococcus aureus,Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcusagalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Chlamydiatracomatis, Neisseria gonorrhoeae, Syphilis (Treponema pallidum),Ureaplasma urealyticum, Mycoplasma genitalium, and/or Gardnerellavaginalis. Also, as a non-limiting example, the target nucleic acid ofinterest could be one or more nucleic acid molecules from a virus, suchas adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E,coronavirus OC43, severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), human metapneumovirus, human rhinovirus, enterovirus,influenza A, influenza A/H1, influenza A/H3, influenza A/H1-2009,influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenzavirus 3, parainfluenza virus 4, respiratory syncytial virus, herpessimplex virus 1, herpes simplex virus 2, human immunodeficiency virus(HIV), human papillomavirus, hepatitis A virus (HAV), hepatitis B virus(HBV), hepatitis C virus (HCV), and/or human parvovirus B19 (B19V).Also, as a non-limiting example, the target nucleic acid of interestcould be one or more nucleic acid molecules from a fungus, such asCandida albicans, Candida auris, Candida glabrata, Candida krusei,Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans,and/or Cryptococcus gattii. As another non-limiting example, the targetnucleic acid of interest could be one or more nucleic acid moleculesfrom a protozoan, such as Trichomonas vaginalis. See, e.g., Table 1 foran exemplary list of human pathogens, Table 2 for an exemplary list ofhuman sexually transmissible diseases.

TABLE 1 Human Pathogens NCBI Taxonomy NCBI Sequence ID Name Category IDNumber Acinetobacter baumannii Bacteria 470 GCF_008632635.1Acinetobacter calcoaceticus Bacteria 471 GCF_002055515.1 AcinetobacterBacteria 909768 Not applicable calcoaceticus-baumannii complex AnaplasmaBacteria 948 GCF_000439775.1 phagocytophilum Bacillus anthracis Bacteria1392 GCF_000008445.1 Bacteroides fragilis Bacteria 817 GCF_016889925.1Bartonella henselae Bacteria 38323 GCF_000612965.1 Bordetellaparapertussis Bacteria 519 GCF_004008295.1 Bordetella pertussis Bacteria520 GCF_004008975.1 Borrelia mayonii Bacteria 1674146 GCF_001936295.1Borrelia miyamotoi Bacteria 47466 GCF_003431845.1 Brucella abortusBacteria 235 GCF_000054005.1 Brucella melitensis Bacteria 29459GCF_000007125.1 Brucella suis Bacteria 29461 GCF_000007505.1Burkholderia mallei Bacteria 13373 GCF_002346025.1 Burkholderiapseudomallei Bacteria 28450 GCF_000756125.1 Campylobacter jejuniBacteria 197 GCF_000009085.1 Chlamydia pneumoniae Bacteria 83558GCF_000007205.1 Chlamydia psittaci Bacteria 83554 GCF_000204255.1Chlamydia Tracomatis Bacteria 813 GCF_000008725.1 Clostridium botulinumBacteria 1491 GCF_000063585.1 Clostridium perfringens Bacteria 1502GCF_020138775.1 Coxiella burnetii Bacteria 777 GCF_000007765.2 Ehrlichiachaffeesis Bacteria 945 GCF_000632965.1 Ehrlichia ewingii Bacteria 947Not available Ehrlichia ruminantium Bacteria 779 GCF_013460375.1Enterobacter cloacae Bacteria 550 GCF_000770155.1 Enterobacter cloacaeBacteria 354276 Not applicable complex Enterococcus faecalis Bacteria1351 GCF_000393015.1 Enterococcus faecium Bacteria 1352 GCF_009734005.1Escherichia coli Bacteria 562 GCF_000008865.2 Francisella tularensisBacteria 263 GCF_000156415.1 Gardnerella vaginalis Bacteria 2702GCF_002861965.1 Haemophilus influenzae Bacteria 727 GCF_000931575.1Klebsiella aerogenes Bacteria 548 GCF_007632255.1 Klebsiella oxytocaBacteria 571 GCF_003812925.1 Klebsiella pneumoniae Bacteria 573GCF_000240185.1 Legionella pneumophila Bacteria 446 GCF_001753085.1Leptospira interrogans Bacteria 173 GCF_002073495.2 Leptospirakirschneri Bacteria 29507 GCF_000243695.2 Leptospira wolffii Bacteria409998 GCF_004770635.1 Listeria monocytogenes Bacteria 1639GCF_000196035.1 Moraxella catarrhalis Bacteria 480 GCF_002080125.1Mycobacterium tuberculosis Bacteria 1773 GCF_000195955.2 Mycoplasmagenitalium Bacteria 2097 GCF_000027325.1 Mycoplasma pneumoniae Bacteria2104 GCF_900660465.1 Neisseria gonorrhoeae Bacteria 485 GCF_013030075.1Neisseria meningitidis Bacteria 487 GCF_008330805.1 Proteus hauseriBacteria 183417 GCF_004116975.1 Proteus mirabilis Bacteria 584GCF_000069965.1 Proteus penneri Bacteria 102862 GCF_022369495.1 Proteusvulgaris Bacteria 585 GCF_000754995.1 Pseudomonas aeruginosa Bacteria287 GCF_000006765.1 Rickettsia parkeri Bacteria 35792 GCF_005549115.1GCA_018610945.1 GCF_000965075.1 GCF_000965085.1 GCF_000284195.1GCF_000965145.1 Rickettsia prowazekii Bacteria 782 GCF_000277165.1Rickettsia rickettsii Bacteria 783 GCF_000017445.4 Salmonella bongoriBacteria 54736 GCF_000439255.1 Salmonella enterica Bacteria 28901GCF_000006945.2 Salmonella enterica Bacteria 28901 GCF_000006945.2Serratia marcescens Bacteria 615 GCF_003516165.1 Shigella boydiiBacteria 621 GCF_001905915.1 Shigella dysenteriae Bacteria 622GCF_001932995.2 Shigella flexneri Bacteria 623 GCF_000006925.2 Shigellasonnei Bacteria 624 GCF_013374815.1 Staphylococcus auerus Bacteria 1280GCF_000013425.1 Staphylococcus enterotoxin B Bacteria 1280 U93688.2Staphylococcus epidermidis Bacteria 1282 GCF_006094375.1 Staphylococcuslugdunensis Bacteria 28035 GCF_001558775.1 Stenotrophomonas Bacteria40324 GCF_900475405.1 maltophilia Streptococcus agalactiae Bacteria 1311GCF_001552035.1 Streptococcus pneumoniae Bacteria 1313 GCF_002076835.1Streptococcus pyogenes Bacteria 1314 GCF_900475035.1 Treponema pallidumBacteria 160 GCF_000246755.1 Ureaplasma urealyticum Bacteria 2130GCF_000021265.1 Vibrio parahaemolyticus Bacteria 670 GCF_000196095.1Vibrio vulnificus Bacteria 672 GCF_002204915.1 Yersinia enterocoliticaBacteria 630 GCF_001160345.1 Yersinia pestis Bacteria 632GCF_000222975.1 Candida albicans Fungus 5476 GCF_000182965.3 Candidaauris Fungus 498019 GCF_002775015.1 Candida glabrata Fungus 5478GCF_000002545.3 Candida parapsilosis Fungus 5480 GCF_000182765.1 Candidatropicalis Fungus 5482 GCF_000006335.3 Coccidioides immitis Fungus 5501GCF_000149335.2 Coccidioides posadasii Fungus 199306 GCF_000151335.2Cokeromyces recurvatus Fungus 90255 GCA_000697235.1 Cryptococcus gattiiFungus 37769 GCF_000185945.1 Cryptococcus neoformans Fungus 5207GCF_000091045.1 Cunninghamella Fungus 90251 GCA_000697215.1bertholletiae Encephalitozoon cuniculi Fungus 6035 GCF_000091225.1Encephalitozoon hellem Fungus 27973 GCF_000277815.2 Encephalitozoonintestinalis Fungus 58839 GCF_000146465.1 Enterocystozoon bieneusiFungus 31281 GCF_000209485.1 Mortierella wolfii Fungus 90253GCA_016098105.1 Pichia kudriavzevii Fungus 4909 GCF_003054445.1Saksenaea vasiformis Fungus 90258 GCA_000697055.1 Syncephalastrum Fungus13706 GCA_002105135.1 racemosum Trichomonas vaginalis Fungus 5722GCF_000002825.2 Ricinus communis Plant 3988 GCF_019578655.1 Acanthamoebacastellanii Protozoa 5755 GCF_000313135.1 Babesia divergens Protozoa32595 GCA_001077455.2 Babesia microti Protozoa 5868 GCF_000691945.2Balamuthia mandrillaris Protozoa 66527 GCA_001185145.1 Cryptosporidiumparvum Protozoa 5807 GCF_000165345.1 Cyclospora cayatanensis Protozoa88456 GCF_002999335.1 Entamoeba histolytica Protozoa 5759GCF_000208925.1 Giardia lamblia Protozoa 5741 GCF_000002435.2 Naegleriafowleri Protozoa 5763 GCF_008403515.1 Toxoplasma gondii Protozoa 5811GCF_000006565.2 Alkhumra hemorrhagic Virus 172148 JF416961.1 fever virusArgentinian Virus 2169991 GCF_000856545.1 mammarenavirus Betacoronavirus1 Virus 694003 GCF_000862505.1 GCF_003972325.1 Black Creek Canal Virus1980460 GCF_002817355.1 orthohantavirus California encephalitis Virus1933264 GCF_003972565.1 orthobunyavirus Chapare mammarenavirus Virus499556 GCF_000879235.1 Chikungunya virus Virus 37124 GCF_000854045.1Crimean-Congo Virus 1980519 GCF_000854165.1 hemorrhagic feverorthnairovirus Dabie bandavirus Virus 2748958 GCF_000897355.1GCF_003087855.1 Deer tick virus Virus 58535 MZ148230 to MZ148271 Denguevirus 1 Virus 11053 GCF_000862125.1 Dengue virus 2 Virus 11060GCF_000871845.1 Dengue virus 3 Virus 11069 GCF_000866625.1 Dengue virus4 Virus 11070 GCF_000865065.1 Eastern equine encephalitis Virus 11021GCF_000862705.1 virus Enterovirus A Virus 138948 GCF_002816655.1GCF_000861905.1 GCF_001684625.1 Enterovirus B Virus 138949GCF_002816685.1 GCF_000861325.1 Enterovirus C Virus 138950GCF_000861165.1 Enterovirus D Virus 138951 GCF_000861205.1GCF_002816725.1 Guanarito mammarenavirus Virus 45219 GCF_000853765.1Heartland bandavirus Virus 2747342 GCF_000922255.1 Hendra henipavirusVirus 63330 GCF_000852685.1 Hepacivirus C Virus 11103 GCF_002820805.1GCF_000861845.1 GCF_000871165.1 GCF_000874285.1 GCF_001712785.1hepatitis A virus Virus 208726 K02990.1 M14707.1 M20273.1 X75215.1AB020564.1 hepatitis B virus Virus 10407 GCF_000861825.2 hepatitis Cvirus Virus 11103 GCF_002820805.1 GCF_000861845.1 GCF_000871165.1GCF_000874285.1 GCF_000874265.1 GCF_001712785.1 Hepatovirus A Virus12092 GCF_000860505.1 Human adenovirus A Virus 129875 GCF_000846805.1Human adenovirus B Virus 108098 GCF_000857885.1 Human adenovirus C Virus129951 GCF_000858645.1 Human adenovirus D Virus 130310 GCF_000885675.1Human adenovirus E Virus 130308 GCF_000897015.1 Human adenovirus F Virus130309 GCF_000846685.1 Human adenovirus G Virus 536079 GCF_000847325.1Human alphaherpesvirus 1 Virus 10298 GCF_000859985.2 Humanalphaherpesvirus 2 Virus 10310 GCF_000858385.2 human betaherpesvirus 6AVirus 32603 GCF_000845685.2 human betaherpesvirus 6B Virus 32604GCF_000846365.1 Human coronavirus 229E Virus 11137 GCF_001500975.1GCF_000853505.1 Human coronavirus HKU1 Virus 290028 GCF_000858765.1Human coronavirus NL63 Virus 277944 GCF_000853865.1 Human coronavirusOC43 Virus 31631 GCF_003972325.1 Human gammaherpesvirus 8 Virus 37296GCF_000838265.1 Human immunodeficiency virus 1 Virus 11676GCF_000864765.1 Human immunodeficiency virus 2 Virus 11709GCF_000856385.1 human metapneumovirus Virus 162145 GCF_002815375.1 humanpapillomavirus Virus GCF_001274345.1 Human polyomavirus 1 Virus 1891762GCF_000837865.1 Human polyomavirus 2 Virus 1891763 GCF_000863805.1 humanrhinovirus A Virus 147711 GCF_000862245.1 GCF_002816835.1 humanrhinovirus B Virus 147712 GCF_000861265.1 GCF_002816855.1 humanrhinovirus C Virus 463676 GCF_002816885.1 GCF_000872325.1 Influenza Avirus Virus 11320 GCF_001343785.1 GCF_000851145.1 GCF_000866645.1Influenza B virus Virus 11520 GCF_000820495.2 Influenza C virus Virus11552 GCF_000856665.10 Influenza D virus Virus 1511084 GCF_002867775.1Japanese encephalitis virus Virus 11072 GCF_000862145.1 Kyasanur Forestdisease virus Virus 33743 GCF_002820625.1 La Crosse orthobunyavirusVirus 2560547 GCF_000850965.1 Lassa virus Virus 11620 GCF_000851705.1Lujo mammarenavirus Virus 649188 GCF_000885555.1 Lyssavirus australisVirus 90961 GCF_000850325.1 Marburg virus Virus NC_001608.3 Measlesmorbillivirus Virus 11234 GCF_000854845.1 Middle East respiratory Virus1335626 GCF_002816195.1 syndrome-related GCF_000901155.1 coronavirusMonongahela hantavirus Virus 2259728 MH539865 MH539866 MH539867 New Yorkhantavirus Virus 44755 U36803.1 U36802.1 U36801.1 U09488.1 Nipahhenipavirus Virus 121791 GCF_000863625.1 Norwalk virus Virus 11983GCF_000864005.1 GCF_008703965.1 GCF_008703985.1 GCF_008704025.1GCF_010478905.1 GCF_000868425.1 Omsk hemorrhagic fever virus Virus 12542GCF_000855505.1 parainfluenza virus 1 Virus 12730 GCF_000848705.1NC_003461 parainfluenza virus 2 Virus X57559.1 AF533010 AF533011AF533012 parainfluenza virus 3 Virus 11216 GCA_006298365.1GCA_000850205.1 parainfluenza virus 4 Virus 2560526 NC_021928.1Paslahepevirus balayani Virus 1678141 GCF_000861105.1 Poliovirus Virus138950 GCF_000861165.1 Primate erythroparvovirus 1 Virus 1511900GCF_000839645.1 Rabies lyssavirus Virus 11292 GCF_000859625.1respiratory syncytial virus Virus 12814 GCF_000856445.1 Rift Valleyvirus Virus 11588 HE687302 HE687307 Saint Louis encephalitis Virus 11080GCF_000866785.1 virus Sapporo virus Virus 95342 GCF_000849945.1GCF_000855765.1 GCF_000854265.1 GCF_001008475.1 GCF_000853825.1SARS-related coronavirus Virus 694009 GCF_000864885.1 GCF_009858895.2Severe acute respiratory Virus 2901879 NC_004718.3 syndrome coronavirus1 Severe acute respiratory Virus 2697049 NC_045512.2 syndromecoronavirus 2 Sin Nombre virus Virus 1980491 GCF_000854765.1 Tick-borneencephalitis virus Virus 11084 GCF_000863125.1 Variola major Virus 12870not available Variola minor Virus 53258 not available Variola virusVirus 10255 GCF_000859885.1 Venezuelan equine Virus 11036GCF_000862105.1 encephalitis virus West Nile virus Virus 11082GCF_000861085.1 GCF_000875385.1 Western equine encephalitis virus Virus11039 GCF_000850885.1 Yellow fever virus Virus 11089 GCF_000857725.1Zaire ebolavirus Virus 186538 GCF_000848505.1 Zika virus Virus 64320GCF_000882815.3 GCF_002366285.1

TABLE 2 Human STD pathogens NCBI Taxonomy NCBI Sequence Name Category IDID Number Pthirus pubis Animal 121228 MT721740.1 Sarcoptes scabieiAnimal 52283 GCA_020844145.1 Chlamydia trachomatis Bacteria 813GCF_000008725.1 Gardnerella vaginalis Bacteria 2702 GCF_002861965.1Haemophilus ducreyi Bacteria 730 GCF_001647695.1 Mycoplasma genitaliumBacteria 2097 GCF_000027325.1 Neisseria gonorrhoeae Bacteria 485GCF_013030075.1 Treponema pallidum Bacteria 160 GCF_000246755.1Trichomonas vaginalis Protozoa 5722 GCF_000002825.2 Hepacivirus C Virus11103 GCF_002820805.1 Hepatitis B virus Virus 10407 GCF_000861825.2Hepatitis delta virus Virus 12475 GCF_000856565.1 Hepatovirus A Virus12092 GCF_000860505.1 Human alphaherpesvirus 1 Virus 10298GCF_000859985.2 Human immunodeficiency Virus 11676 GCF_000864765.1 virus1 Human immunodeficiency Virus 11709 GCF_000856385.1 virus 2 Humanpapillomavirus Virus 10566 GCF_001274345.1

Additionally, the target nucleic acid of interest may originate in anorganism such as a bacterium, virus, fungus or other pest that infectslivestock or agricultural crops. Such organisms include avian influenzaviruses, mycoplasma and other bovine mastitis pathogens, Clostridiumperfringens, Campylobacter sp., Salmonella sp., Pospirivoidae,Avsunvirodiae, Panteoea stewartii, Mycoplasma genitalium, Sprioplasmasp., Pseudomonas solanacearum, Erwinia amylovora, Erwinia carotovora,Pseudomonas syringae, Xanthomonas campestris, Agrobacterium tumefaciens,Spiroplasma citri, Phytophthora infestans, Endothia parasitica,Ceratocysis ulmi, Puccinia graminis, Hemilea vastatrix, Ustilage maydis,Ustilage nuda, Guignardia bidwellii, Uncinula necator, Botrytiscincerea, Plasmopara viticola, or Botryotinis fuckleina. See, e.g.,Table 3 for an exemplary list of non-human animal pathogens.

TABLE 3 Animal Pathogens NCBI Name Category Taxonomy ID NCBI Sequence IDNumber Acarapis woodi Animal 478375 GCA_023170135.1 Aethina tumidaAnimal 116153 GCF_001937115.1 Chorioptes bovis Animal 420257 Chrysomyabezziana Animal 69364 Cochliomyia hominivorax Animal 115425GCA_004302925.1 Echinococcus granulosus Animal 6210 GCF_000524195.1Echinococcus Animal 6211 GCA_000469725.3 multilocularis Gyrodactylussalaris Animal 37629 GCA_000715275.1 Psoroptes ovis Animal 83912GCA_002943765.1 Sarcoptes scabiei Animal 52283 GCA_020844145.1 Taeniasolium Animal 6204 GCA_001870725.1 Trichinella britovi Animal 45882GCA_001447585.1 Trichinella nativa Animal 6335 GCA_001447565.1Trichinella nelsoni Animal 6336 GCA_001447455.1 Trichinella papuaeAnimal 268474 GCA_001447755.1 Trichinella pseudospiralis Animal 6337GCA_001447645.1 Trichinella spiralis Animal 6334 GCF_000181795.1Trichinella zimbabwensis Animal 268475 GCA_001447665.1 Tropilaelapsclareae Animal 208209 Tropilaelaps koenigerum Animal 208208 Tropilaelapsmercedesae Animal 418985 GCA_002081605.1 Tropilaelaps thaii Animal418986 Varroa destructor Animal 109461 GCF_002443255.1 Varroa jacobsoniAnimal 62625 GCF_002532875.1 Varroa rindereri Animal 109259 Varroaunderwoodi Animal 109260 Anaplasma centrale Bacteria 769 GCF_000024505.1Anaplasma marginale Bacteria 770 GCF_000020305.1 Bacillus anthracisBacteria 1392 GCF_000008445.1 Brucella abortus Bacteria 235GCF_000054005.1 Brucella melitensis Bacteria 29459 GCF_000007125.1Brucella ovis Bacteria 236 GCF_000016845.1 Brucella suis Bacteria 29461GCF_000007505.1 Burkholderia mallei Bacteria 13373 GCF_002346025.1Burkholderia pseudomallei Bacteria 28450 GCF_000756125.1 Campylobacterfetus Bacteria 196 GCF_000015085.1 Candidatus Xenohaliotis Bacteria84677 californiensis Candidatus Hepatobacter Bacteria 1274402GCF_000742475.1 penaei Chlamydia abortus Bacteria 83555 GCF_900416725.2Chlamydia psittaci Bacteria 83554 GCF_000204255.1 CorynebacteriumBacteria 1719 GCF_001865765.1 pseudotuberculosis Coxiella burnetiiBacteria 777 GCF_000007765.2 Ehrlichia ruminantium Bacteria 779GCF_013460375.1 Francisella tularensis Bacteria 263 GCF_000156415.1Melissococcus plutonius Bacteria 33970 GCF_003966875.1 Mycobacteriumavium Bacteria 1764 GCF_000696715.1 Mycobacterium Bacteria 1773GCF_000195955.2 tuberculosis Mycoplasma capricolum Bacteria 2095GCF_000012765.1 Mycoplasma gallisepticum Bacteria 2096 GCF_000286675.1Mycoplasma mycoides Bacteria 2102 GCF_000023685.1 Mycoplasmaputrefaciens Bacteria 2123 GCF_900476175.1 Mycoplasmopsis agalactiaeBacteria 2110 GCF_009150585.1 Mycoplasmopsis synoviae Bacteria 2109GCF_013393745.1 Paenibacillus larvae Bacteria 1464 GCF_002951935.1Pasteurella multocida Bacteria 747 GCF_000006825.1 Salmonella entericaBacteria 28901 GCF_000006945.2 Streptococcus equi Bacteria 1336GCF_015689455.1 Taylorella equigenitalis Bacteria 29575 GCF_002288025.1Vibrio parahaemolyticus Bacteria 670 GCF_000196095.1 BatrachochytriumFungi 109871 GCF_000203795.1 dendrobatidis Batrachochytrium Fungi1357716 GCA_021556675.1 salamandrivorans Aphanomyces astaci Oomycota112090 GCF_000520075.1 Aphanomyces invadans Oomycota 157072GCF_000520115.1 Babesia bigemina Protozoa 5866 GCF_000981445.1 Babesiabovis Protozoa 5865 GCA_000165395.2 Babesia caballi Protozoa 5871Bonamia exitiosa Protozoa 362532 Bonamia ostreae Protozoa 126728Leishmania amazonensis Protozoa 5659 GCA_005317125.1 Leishmaniabraziliensis Protozoa 5660 GCF_000002845.2 Leishmania donovani Protozoa5661 GCF_000227135.1 Leishmania infantum Protozoa 5671 GCF_000002875.2Leishmania major Protozoa 5664 GCF_000002725.2 Leishmania mexicanaProtozoa 5665 GCF_000234665.1 Leishmania tropica Protozoa 5666GCA_014139745.1 Marteilia refringens Protozoa 107386 Perkinsus marinusProtozoa 31276 GCF_000006405.1 Perkinsus olseni Protozoa 32597GCA_013115135.1 Theileria annulata Protozoa 5874 GCF_000003225.4Theileria equi Protozoa 5872 GCF_000342415.1 Theileria parva Protozoa5875 GCF_000165365.1 Tritrichomonas foetus Protozoa 1144522GCA_001839685.1 Trypanosoma brucei Protozoa 5691 GCF_000002445.2Trypanosoma congolense Protozoa 5692 GCA_002287245.1 Trypanosomaequiperdum Protozoa 5694 GCA_001457755.2 Trypanosoma evansi Protozoa5697 GCA_917563935.1 Trypanosoma vivax Protozoa 5699 GCA_021307395.1African horse Virus 40050 GCF_000856125.1 sickness virus African swinefever virus Virus 10497 GCF_000858485.1 Akabane orthobunyavirus Virus1933178 GCF_000871205.1 Alcelaphine Virus 35252 GCF_000838825.1gammaherpesvirus 1 Alphaarterivirus equid Virus 2499620 GCF_000860865.1Alphacoronavirus 1 Virus 693997 GCF_000856025.1 Ambystoma tigrinum virusVirus 265294 GCF_000841005.1 Avian coronavirus Virus 694014GCF_012271565.1 Avian influenza virus Virus 11309 Avian metapneumovirusVirus 38525 GCF_002989735.1 Avian orthoavulavirus 1 Virus 2560319GCF_002834085.1 Avihepatovirus A Virus 691956 GCF_000869945.1Betaarterivirus suid 1 Virus 2499680 GCF_003971765.1 Bluetongue virusVirus 40051 GCF_000854445.3 Bovine alphaherpesvirus 1 Virus 10320GCF_008777455.1 Bovine leukemia virus Virus 11901 GCF_000853665.1Camelpox virus Virus 28873 GCF_000839105.1 Caprine arthritis Virus 11660GCF_000857525.1 encephalitis virus Crimean-Congo Virus 1980519GCF_000854165.1 hemorrhagic fever orthonairovirus Cyprinid herpesvirus 3Virus 180230 GCF_000871465.1 Decapod iridescent virus 1 Virus 2560405GCF_004788555.1 Decapod Virus 1513224 GCF_000844705.1 penstyldensovirus1 Deformed wing virus Virus 198112 GCF_000852585.1 Eastern equine Virus11021 GCF_000862705.1 encephalitis virus Epizootic haematopoietic Virus100217 GCF_001448375.1 necrosis virus Epizootic hemorrhagic Virus 40054GCF_000885335.1 disease virus Equid alphaherpesvirus 1 Virus 10326GCF_000844025.1 Equid alphaherpesvirus 4 Virus 10331 GCF_000846345.1Equine infectious Virus 11665 GCF_000847605.1 anemia virusFoot-and-mouth disease Virus 12110 GCF_002816555.1 virus Frog virus 3Virus 10493 GCF_002826565.1 Gallid alphaherpesvirus 1 Virus 10386GCF_000847005.1 Goatpox virus Virus 186805 GCF_000840165.1 Haliotidherpesvirus 1 Virus 1513231 GCF_000900375.1 Hendra henipavirus Virus63330 GCF_000852685.1 Infectious bursal Virus 10995 GCF_000855485.1disease virus Infectious spleen Virus 180170 GCF_000848865.1 and kidneynecrosis virus Influenza A virus Virus 11320 GCF_000851145.1 Isavirussalaris Virus 55987 GCF_000854145.2 Japanese encephalitis virus Virus11072 GCF_000862145.1 Lumpy skin disease virus Virus 59509GCF_000839805.1 Lyssavirus rabies Virus 11292 GCF_000859625.1Macrobrachium Virus 222557 GCA_000856985.1 rosenbergii nodavirus MiddleEast respiratory Virus 1335626 GCF_002816195.1 syndrome-relatedcoronavirus Myxoma virus Virus 10273 GCF_000843685.1 Nairobi sheep Virus1980526 GCF_002117695.1 disease orthonairovirus Nipah henipavirus Virus121791 GCF_000863625.1 Norwegian salmonid Virus 344701 alphavirusNovirhabdovirus piscine Virus 1980916 GCF_000856505.1 Novirhabdovirussalmonid Virus 1980917 GCF_000850065.1 Penaeid shrimp infectious Virus282786 GCA_000866305.1 myonecrosis virus Peste des petits ruminantsVirus 2593991 GCF_000866445.1 virus Pestivirus C Virus 2170082GCF_000864685.1 GCF_003034095.1 Pestivirus A Virus 2170080GCF_000861245.1 Rabbit hemorrhagic Virus 11976 GCF_000861285.1 diseasevirus Rift Valley fever Virus 1933187 GCF_000847345.1 phlebovirusRinderpest morbillivirus Virus 11241 GCF_000856645.1 Severe acute Virus694009 GCF_000864885.1 respiratory syndrome- related coronavirusSheeppox virus Virus 10266 GCF_000840205.1 Slow bee paralysis virusVirus 458132 GCF_000887395.1 Sprivirus cyprinus Virus 696863GCF_000850305.1 Suid alphaherpesvirus 1 Virus 10345 GCF_000843825.1Swine vesicular Virus 12075 disease virus Taura syndrome virus Virus142102 GCF_000849385.1 Tilapinevirus tilapiae Virus 2034996GCF_001630085.1 Venezuelan equine Virus 11036 GCF_000862105.1encephalitis virus Vesiculovirus indiana Virus 1972577 GCF_000850045.1Visna-maedi virus Virus 2169971 GCF_000849025.1 West Nile Virus Virus11082 GCF_000861085.1 Western equine Virus 11039 GCF_000850885.1encephalitis virus White spot syndrome virus Virus 342409GCF_000848085.2 Yellow head virus Virus 96029 GCF_003972805.1

In some embodiments, other target nucleic acids of interest may be fornon-infectious conditions, e.g., to be used for genotyping, includingnon-invasive prenatal diagnosis of, e.g, trisomies, other chromosomalabnormalities, and known genetic diseases such as Tay Sachs disease andsickle cell anemia. Other target nucleic acids of interest and samplesare described herein, such as human biomarkers for cancer. An exemplarylist of human biomarkers is in Table 4. Target nucleic acids of interestmay include engineered biologics, including cells such as CAR-T cells,or target nucleic acids of interest from very small or rare samples,where only small volumes are available for testing.

TABLE 4 Human Biomarkers NCBI NCBI Taxonomy Gene Biomarker DiseaseSample ID ID Aβ42, amyloid beta- Alzheimer disease CSF 9606 351 proteinprion protein Alzheimer disease, prion CSF 9606 5621 disease Vitamin Dbinding multiple sclerosis CSF 9606 2638 protein progression CXCL13multiple sclerosis CSF 9606 10563 alpha-synuclein parkinsonian disordersCSF 9606 6622 tau protein parkinsonian disorders CSF 9606 4137 Apo IIparkinsonian disorders CSF 9606 336 ceruloplasmin parkinsonian disordersCSF 9606 1356 peroxisome parkinsonian disorders CSF 9606 5467proliferation- activated PD receptor parkin neurogenerative CSF 96065071 disorders PTEN induced neurogenerative CSF 9606 65018 putativekinase I disorders DJ-1 (PARK7) neurogenerative CSF 9606 11315 disordersleucine-rich repeat neurogenerative CSF 9606 120892 kinase disorderssecretogranin II bipolar disorder CSF 9606 7857 neurofilament lightaxonal degeneration CSF 9606 4747 chain IL-12B, CXDL13, Intrathecalinflammation CSF 9606 3593, 10563, IL-8 3576 ACE2 cardiovascular diseaseblood 9606 59272 alpha-amylase cardiovascular disease saliva 9606 276alpha-feto protein pregnancy blood 9606 174 albumin urine diabetes 9606213 albumin, urea albuminuria urine 9606 213 neutrophil gelatinase-acute kidney injury urine 9606 3934 associated lipocalin (NGAL) IL-18acute kidney injury urine 9606 3606 liver fatty acid acute kidney injuryurine 9606 2168 binding protein Dkk-3 prostate cancer semen 9606 27122autoantibody to early diagnosis blood 9606 CD25 esophageal squamous cellcarcinoma hTERT lung cancer blood 9606 7015 CA125 (MUC16) lung cancerblood 9606 94025 VEGF lung cancer blood 9606 7422 IL-2 lung cancer blood9606 3558 osteopontin lung cancer blood 9606 6696 BRAF, CCNI, EGRF, lungcancer saliva 9606 673, 16007, FGF19, FRS2, 1956, 9965, GREB1, and LZTS110818, 9687, 11178 human epididymis ovarian cancer blood 9606 10406protein 4 CA125 ovarian cancer saliva 9606 94025 EMP1 nasopharyngealsaliva 9606 13730 carcinoma IL-8 oral cancer saliva 9606 3576carcinoembryonic oral or salivary saliva 9606 1048 antigen malignanttumors thioredoxin Spinalcellular carcinoma saliva 9606 7295 AIP (arylAcute intermittent blood 9606 9049 hydrocarbon receptor porphyria,somatotroph interacting protein) adenoma, prolactin- producing pituitarygland adenoma ALK receptor Neuroblastoma blood 9606 238 tyrosine kinasesusceptibility, large cell lymphoma BAP1 (BRCA1 BAP1-related tumor blood9606 8314 associated protein 1) predisposition, melanoma susceptibilityBLM Bloom syndrome blood 9606 641 BRCA1 Breast-ovarian cancer blood 9606672 susceptibility, familial breast cancer BRCA2 Breast-ovarian cancerblood 9606 675 susceptibility, familial breast cancer, gliomasusceptibility CASR (calcium Epilepsy susceptibility blood 9606 846sensing receptor) CDC73 Hyperparathyroidism 2 blood 9606 79577 with jawtumors CEBPA Acute myloid leukemia blood 9606 1050 EPCAM Colorectalcancer blood 9606 4072 FH hypercholesterolemia blood 9606 2271 GATA2Acute myeloid leukemia blood 9606 2642 MITF Melanoma susceptibilityblood 9606 4286 MSH2 Lynch syndrome blood 9606 4436 MSH3 Endometrialcarcinoma blood 9606 4437 MSH6 Endometrial carcinoma, blood 9606 2956colorectal cancer NF1 Neurofibromatosis, blood 9606 4763 juvenilemyelomonocytic leukemia PDGRA Eosinophilic leukemia, blood 9606 5156recurrent inflammatory gastrointestinal fibroids PHOX2B Neuroblastomablood 9606 8929 susceptibility POT1 Melanoma blood 9606 25913susceptibility, glioma susceptibility

The target nucleic acids of interest may be taken from environmentalsamples. A list of exemplary biosafety pathogens is in Table 5, and anexemplary list of known viruses is in Table 6.

TABLE 5 Exemplary Laboratory Biosafety Parasites and Pathogens NCBI NCBITaxonomy Taxonomy Name Category ID Name Category ID Acarapis woodiAnimal 478375 Streptococcus Bacteria 1349 uberis Aethina tumida Animal116153 Besnoitia besnoiti Chromista 94643 Alaria americana Animal2282137 Bonamia exitiosa Chromista 362532 Amblyomma Animal 6943 Bonamiaostreae Chromista 126728 americanum Amblyomma Animal 34609 AmniculicolaFungus 2566060 maculatum longissima Amphimerus Animal Arthroderma Fungus1592210 pseudofelineus amazonicum Ancylostoma Animal 369059 AschersoniaFungus 370936 braziliense hypocreoidea Ancylostoma Animal 29170Aspergillago Fungus 41064 caninum clavatoflava Ancylostoma Animal 51022Aspergillus Fungus 1904037 duodenale acidohumus Anisakis Animal 303229Aspergillus acidus Fungus 1069201 pegreffii Anisakis simplex Animal 6269Aspergillus Fungus 487661 aculeatinus Baylisascaris Animal 575210Aspergillus Fungus 5053 columnaris aculeatus Baylisascaris AnimalAspergillus aeneus Fungus 41754 melis Baylisascaris Animal 6259Aspergillus affinis Fungus 1070780 procyonis Bunostomum Animal 577651Aspergillus Fungus 657433 phlebotomum alabamensis Ceratonova Animal60662 Aspergillus Fungus 209559 shasta alliaceus Chrysomya Animal 69364Aspergillus Fungus 710228 bezziana amazonicus Cochliomyia Animal 115425Aspergillus Fungus 176160 hominivorax ambiguus Dicrocoelium Animal 57078Aspergillus Fungus 1220191 dendriticum amoenus Diphyllobothrium Animal28845 Aspergillus Fungus 296546 dendriticum amyloliquefaciensDiphyllobothrium Animal 60516 Aspergillus Fungus 176161 latum amylovorusEchinococcus Animal Aspergillus Fungus 2783700 granulosa angustatusEchinococcus Animal 6211 Aspergillus Fungus 454240 multilocularisanomalus Echinococcus Animal 6212 Aspergillus Fungus 37233 oligarthrusanthodesmis Echinococcus Animal 260967 Aspergillus Fungus 478867shiquicus apicalis Echinococcus Animal 6213 Aspergillus Fungus 1140386vogeli appendiculatus Echinostoma Animal 1873862 Aspergillus Fungus656916 cinetorchis arachidicola Echinostoma Animal 48216 AspergillusFungus 1458899 hortense ardalensis Echinostoma liei Animal 48214Aspergillus arvii Fungus 368784 Echinostoma Animal 48217 AspergillusFungus 1695225 revolutum askiburgiensis Fasciola hepatica Animal 6192Aspergillus Fungus 176163 asperescens Fascioloides Animal 394415Aspergillus Fungus 1245746 magna assulatus Gyrodactylus Animal 37629Aspergillus Fungus 1810904 salaris astellatus Ixodes pacificus Animal29930 Aspergillus Fungus 41725 aurantiobrunneus Ixodes ricinus Animal34613 Aspergillus Fungus 2663348 aurantiopurpureus Ixodes scapularisAnimal 6945 Aspergillus Fungus 41755 aureolatus Metagonimus Animal 84529Aspergillus Fungus 41288 yokogawai aureoterreus Metorchis AnimalAspergillus aureus Fungus 309747 conjunctus Myxobolus Animal 59783Aspergillus Fungus 138274 cerebralis auricomus Nanophyetus Animal 240278Aspergillus Fungus 1250384 salmincola australiensis Necator Animal 51031Aspergillus Fungus 1220192 americanus austroafricanus Oestrus ovisAnimal 123737 Aspergillus Fungus 36643 avenaceus Opisthorchis Animal147828 Aspergillus Fungus 105351 felineus awamori Opisthorchis Animal6198 Aspergillus Fungus 2070749 viverrini baarnensis Parafilaria Animal2282233 Aspergillus Fungus 1194636 bovicola baeticus Paragonimus Animal100269 Aspergillus Fungus 522521 kellicotti bahamensis ParagonimusAnimal 59628 Aspergillus Fungus 1226010 miyazakii, bertholletiaeParagonimus Animal 34504 Aspergillus Fungus 176164 westermani biplanusPsoroptes ovis Animal 83912 Aspergillus Fungus 41753 bisporusRhipicephalus Animal 34611 Aspergillus Fungus 109264 annulatus bombycisRhipicephalus Animal 34632 Aspergillus Fungus 1810893 sanguineusbotswanensis Sarcoptes scabiei Animal 52283 Candida albicans Fungus 5476Taenia multiceps Animal 94034 Candida glabrata Fungus 5478 Taeniasaginata Animal 6206 Candida krusei Fungus 4909 Taenia solium Animal6204 Candida Fungus 5480 parapsilosis Toxocara canis Animal 6265 Candidatropicalis Fungus 5482 Toxocara cati Animal 6266 Cryptococcus Fungus37769 gattii Trichinella Animal 6334 Cryptococcus Fungus 5207 spiralisneoformans Trichuris suis Animal 68888 Epidermophyton Fungus 34391floccosum Trichuris Animal 36087 Epidermophyton Fungus 74042 trichiurastockdaleae Trichuris vulpis Animal 219738 Fusarium acaciae FungusTropilaelaps Animal 208209 Fusarium acaciae- Fungus 282272 clareaemearnsii Tropilaelaps Animal 418985 Fusarium acicola Fungus mercedesaeUncinaria Animal 125367 Fusarium Fungus stenocephala acremoniopsisVarroa destructor Animal 109461 Fusarium Fungus acridiorumActinobacillus Bacteria 715 Fusarium acutatum Fungus 78861pleuropneumoniae Aeromonas Bacteria 644 Fusarium Fungus hydrophilaaderholdii Aeromonas Bacteria 645 Fusarium Fungus salmonicida adesmiaeAliarcobacter Bacteria 28197 Fusarium Fungus butzleri aduncisporumAliarcobacter Bacteria 28198 Fusarium aecidii- Fungus cryaerophilustussilaginis Aliarcobacter Bacteria 28200 Fusarium Fungus skirrowiiaeruginosam Anaplasma Bacteria 769 Fusarium Fungus 569394 centraleaethiopicum Anaplasma Bacteria 770 Fusarium affine Fungus marginaleAnaplasma Bacteria 948 Fusarium Fungus phagocytophilum agaricorumBacillus anthracis Bacteria 1392 Fusarium Fungus ailanthinum Bacilluscereus Bacteria 1396 Fusarium Fungus alabamense Bartonella Bacteria38323 Fusarium albedinis Fungus henselae Bibersteinia Bacteria 47735Fusarium albertii Fungus trehalosi Borrelia Bacteria 139 Fusarium Fungusburgdorferi albidoviolaceum Brucella abortus Bacteria 235 Fusariumalbiziae Fungus Brucella canis Bacteria 36855 Fusarium Fungusalbocarneum Brucella Bacteria 29459 Fusarium album Fungus melitensisBrucella ovis Bacteria 236 Fusarium Fungus aleurinum Brucella suisBacteria 29461 Fusarium aleyrodis Fungus Burkholderia Bacteria 13373Fusarium Fungus mallei alkanophilum Burkholderia Bacteria 28450 FusariumFungus pseudomallei allescheri Campylobacter Bacteria 195 FusariumFungus coli allescherianum Campylobacter Bacteria 32019 Fusarium allii-Fungus fetus fetus sativi Campylobacter Bacteria 32020 Trichophytonsimii Fungus 63406 fetus venerealis Campylobacter Bacteria 197Trichophyton Fungus 69891 jejuni soudanense Chlamydia Bacteria 83557Trichophyton Fungus 34387 caviae tonsurans Chlamydia felis Bacteria83556 Trichophyton Fungus 63417 verrucosum Chlamydia Bacteria 83560Trichophyton Fungus 34388 muridarum violaceum Chlamydia Bacteria 85991Ochroma Plant 66662 pecorum pyramidale Chlamydia Bacteria 83558 Babesiabigemina Protozoa 5866 pneumoniae Chlamydia Bacteria 83554 Babesia bovisProtozoa 5865 psittaci Chlamydia suis Bacteria 83559 Babesia divergensProtozoa 32595 Chlamydia Bacteria 813 Babesia jakimovi Protozoatrachomatis Chlamydophilus Bacteria Babesia major Protozoa 127461abortus Clostridium Bacteria 1491 Babesia occultans Protozoa 536930botulinum Clostridium Bacteria 1496 Babesia ovata Protozoa 189622difficile Clostridium Bacteria Cryptosporidium Protozoa 5807 perfringensparvum Types A, B, C, and D Coxiella burnetii Bacteria 777 Eimeriaacervulina Protozoa 5801 Cronobacter Bacteria 28141 Eimeria brunettiProtozoa 51314 sakazakii Ehrlichia canis Bacteria 944 Eimeria maximaProtozoa 5804 Ehrlichia Bacteria 945 Eimeria Protozoa 1431345chaffeensis meleagridis Ehrlichia ewingii Bacteria 947 Eimeria necatrixProtozoa 51315 Ehrlichia ondiri Bacteria Eimeria tenella Protozoa 5802Ehrlichia Bacteria 779 Entamoeba Protozoa 5759 ruminantium histolyticaEscherichia coli Bacteria 562 Giardia duodenalis Protozoa 5741Klebsiella Bacteria 548 Giardia lambia Protozoa aerogenes KlebsiellaBacteria 39824 Histomonas Protozoa 135588 granulomatis meleagridisKlebsiella Bacteria 2058152 Ichthyobodo Protozoa 155203 grimontiinecator Klebsiella Bacteria 2153354 Ichthyophthirius Protozoa 5932huaxiensis multifiliis Klebsiella Bacteria 2042302 Isospora burrowsiProtozoa kielensis Klebsiella Bacteria 1134687 Isospora canis Protozoa1662860 michiganensis Klebsiella Bacteria 223378 Isospora felis Protozoa482539 milletis Klebsiella Bacteria 571 Isospora neorivolta Protozoaoxytoca Klebsiella Bacteria 573 Isospora ohioensis Protozoa 279926pneumoniae Klebsiella Bacteria 1463165 Leishmania Protozoa 5660quasipneumoniae braziliensis Klebsiella Bacteria 2026240 LeishmaniaProtozoa 44271 quasivariicola chagasi Klebsiella Bacteria 223379Leishmania Protozoa 5671 senegalensis infantum Klebsiella Bacteria1641362 Marteilia Protozoa 107386 steroids refringens KlebsiellaBacteria 244366 Mikrocytos Protozoa 195010 variicola mackini Proteusmirabilis Bacteria 584 Perkinsus marinus Protozoa 31276 PseudomonasBacteria 89065 Perkinsus olensi Protozoa abietaniphila PseudomonasBacteria 407029 Sarcocystis cruzi Protozoa 5817 acephalitica PseudomonasBacteria 1912599 Sarcocystis hirsuta Protozoa 61649 acidophilaPseudomonas Bacteria 1302376 Sarcocystis Protozoa 61650 adelgestsugashominis Pseudomonas Bacteria 287 Theileria annulata Protozoa 5874aeruginosa Pseudomonas Bacteria 1387231 Theileria buffei Protozoa aestusPseudomonas Bacteria 46677 Theileria Protozoa 77054 agarici lestoquardiPseudomonas Bacteria Theileria Protozoa 540482 akappageensis luwenshuniPseudomonas Bacteria 43263 Theileria mutans Protozoa 27991 alcaligenesPseudomonas Bacteria 101564 Theileria orientalis Protozoa 68886alcaliphila Pseudomonas Bacteria 37638 Theileria parva Protozoa 5875alginovora Pseudomonas Bacteria Theileria sergenti Protozoa 5877alkanolytica Pseudomonas Bacteria 237609 Theileria Protozoa 507731alkylphenolica uilenbergi Pseudomonas Bacteria 2740531 ToxoplasmaProtozoa 5811 allii gondii Pseudomonas Bacteria 2810613 Trichomonasfetus Protozoa alliivorans Pseudomonas Bacteria 2774460 TrichomonasProtozoa 56777 allokribbensis gallinae Pseudomonas Bacteria 1940621Trichomonas Protozoa 1440121 alloputida stableri Pseudomonas Bacteria2842348 Trypanosoma Protozoa 5691 alvandae brucei Pseudomonas Bacteria47877 Trypanosoma Protozoa 5692 amygdali congolense Pseudomonas Bacteria32043 Trypanosoma Protozoa 5693 amyloderamosa cruzi Pseudomonas Bacteria2710589 Abras virus Virus 2303487 anatoliensis Pseudomonas Bacteria147728 Absettarov virus Virus andersonii Pseudomonas Bacteria 53406 AbuHammad Virus 248058 anguilliseptica virus Pseudomonas Bacteria 219572Abu Mina virus Virus 248059 antarctica Pseudomonas Bacteria 485870 Acadovirus Virus anuradhapurensis Pseudomonas Bacteria 2710591 Acara virusVirus 2748201 arcuscaelestis Pseudomonas Bacteria 289370 Achiote virusVirus 2036702 argentinensis Pseudomonas Bacteria 702115 Adana virusVirus 1611877 arsenicoxydans Pseudomonas Bacteria 2842349 Adelaide RiverVirus 31612 asgharzadehiana virus Pseudomonas Bacteria 2219225 Adriavirus Virus asiatica Pseudomonas Bacteria 53407 Aedes aegypti Virus186156 asplenii densovirus Pseudomonas Bacteria 1190415 Aedes albopictusVirus 35338 asturiensis densovirus Pseudomonas Bacteria 1825787 Aedesflavivirus Virus 390845 asuensis Pseudomonas Bacteria 2565368 Aedesgalloisi Virus 1046551 atacamensis flavivirus Pseudomonas Bacteria2609964 Aedes Virus atagonensis pseudoscutellaris densovirus PseudomonasBacteria 86192 Aedes Virus 341721 aurantiaca pseudoscutellaris reovirusPseudomonas Bacteria 587851 Aedes vexans Virus 7163 aureofaciensPseudomonas Bacteria 46257 African horse Virus 40050 avellanae sicknessvirus Pseudomonas Bacteria 1869229 African swine Virus 10497 aylmerensisfever virus Pseudomonas Bacteria 2843612 Aguacate virus Virus 1006583azadiae Pseudomonas Bacteria Aino virus Virus 11582azerbaijanoccidentalis Pseudomonas Bacteria Akabane virus Virus 70566azerbaijanorientalis Pseudomonas Bacteria 291995 Alajuela virus Virus1552846 azotifigens Pseudomonas Bacteria 47878 Alcelaphine Virus 35252azotoformans gammaherpesvirus 1 Pseudomonas Bacteria 674054 Alenquervirus Virus 629726 baetica Pseudomonas Bacteria 74829 Aleutian MinkVirus balearica Disease Pseudomonas Bacteria 2762576 Alfuy virus Virus44017 baltica Pseudomonas Bacteria 2843610 Alkhumra Virus 172148bananamidigenes hemorrhagic fever virus Pseudomonas Bacteria AllpahuayoVirus 144752 bathycetes mammarenavirus Pseudomonas Bacteria 226910Almeirim virus Virus batumici Pseudomonas Bacteria 556533 AlmendravirusVirus 1972683 benzenivorans arboretum Pseudomonas Bacteria 2681983Almendravirus Virus 1972685 bijieensis cootbay Pseudomonas Bacteria254015 Almpiwar virus Virus 318843 blatchfordae Pseudomonas Bacteria2044872 Alocasia Virus 4456 bohemica macrorrhizos Pseudomonas Bacteria289003 Altamira virus Virus borbori Pseudomonas Bacteria 84586 Amaparivirus Virus borealis Pseudomonas Bacteria 2842352 Ambe virus Virus1926500 botevensis Pseudomonas Bacteria 930166 Amga virus Virus 1511732brassicacearum Pseudomonas Bacteria 2708063 Amur/Soochong Virusbrassicae virus Pseudomonas Bacteria 129817 Anadyr virus Virus 1642852brenneri Pseudomonas Bacteria 2316085 Anajatuba virus Virus 379964bubulae Pseudomonas Bacteria 2731681 Ananindeua virus Virus 1927813campi Pseudomonas Bacteria 915099 Andasibe virus Virus canadensisPseudomonas Bacteria 2859001 Andes Virus 1980456 canavaninivoransorthohantavirus Pseudomonas Bacteria 86840 Anhanga virus Virus 904722cannabina Pseudomonas Bacteria 1495066 Anhembi virus Virus 273355capeferrum Pseudomonas Bacteria 2810614 Anopheles A virus Virus 35307capsici Pseudomonas Bacteria 46678 Anopheles B virus Virus 35308caricapapayae Pseudomonas Bacteria 2487355 Anopheles Virus 2053814carnis flavivirus Pseudomonas Bacteria 1451454 Anopheles Virus 487311caspiana gambiae densovirus Pseudomonas Bacteria 2320867 Antequera virusVirus 2748239 cavernae Pseudomonas Bacteria 2320866 Apoi virus Virus64280 cavernicola Pseudomonas Bacteria 651740 Araguari virus Virus352236 cedrina Pseudomonas Bacteria 155077 Aransas Bay virus Virus1428582 cellulosa Pseudomonas Bacteria 1583341 Araraquara virus Virus139032 cerasi Pseudomonas Bacteria Bluetongue virus Virus 40051chaetocerotis Pseudomonas Bacteria 489632 Bobaya virus Virus 2818228chengduensis Pseudomonas Bacteria 203192 Bobia virus Viruschloritidismutans Pseudomonas Bacteria 587753 Boraceia virus Viruschlororaphis Pseudomonas Bacteria 36746 Borna disease Virus 12455cichorii virus Pseudomonas Bacteria 53408 Botambi virus Viruscitronellolis Pseudomonas Bacteria 416340 Boteke virus Virus 864698clemancea Pseudomonas Bacteria Bouboui virus Virus 64295 coenobiosPseudomonas Bacteria 1605838 Bourbon virus Virus 1618189 coleopterorumPseudomonas Bacteria 658457 Bovine ephemeral Virus 11303 composti fevervirus Pseudomonas Bacteria 200452 Bovine Herpes Virus congelans Virus 1Pseudomonas Bacteria 53409 Bovine leukemia Virus 11901 coronafaciensvirus Pseudomonas Bacteria 47879 Bovine Virus 11246 corrugataorthopneumovirus Pseudomonas Bacteria 168469 Bovine viral Virus 11099costantinii diarrhea virus 1 Pseudomonas Bacteria 157783 Bowe virusVirus 1400425 cremoricolorata Pseudomonas Bacteria 2724178 Bozo virusVirus 273349 cremoris Pseudomonas Bacteria 2697028 Cumuto virus Virus1457166 crudilactis Pseudomonas Bacteria 543360 Cupixi Virus 208899cuatrocienegasensis mammarenavirus Pseudomonas Bacteria 2781239Curionopolis virus Virus 490110 cyclaminis Pseudomonas Bacteria 2487519Cyprinid Virus 180230 daroniae herpesvirus 3 Pseudomonas Bacteria 882211Czech Aedes Virus deceptionensis vexans flavivirus virus PseudomonasBacteria 1876757 D'Aguilar virus Virus defluvii Pseudomonas Bacteria366289 Dabakala virus Virus delhiensis Pseudomonas Bacteria 43306Dabieshan virus Virus 1167310 denitrificans Pseudomonas Bacteria DakNong virus Virus 1238455 diazotrophicus Pseudomonas Bacteria 135830Dakar bat virus Virus 64282 diterpeniphila Pseudomonas Bacteria 1163398Dandenong virus Virus 483046 donghuensis Pseudomonas Bacteria 2487520Dashli virus Virus 1764087 dryadis Pseudomonas Bacteria 459528 Deer tickvirus Virus 58535 duriflava Pseudomonas Bacteria 2006980 Dengue virusVirus 12637 edaphica Pseudomonas Bacteria 2842353 Dengue virus 1 Virusekonensis virus Pseudomonas Bacteria 179878 Cumuto virus Virus 1457166elodea Pseudomonas Bacteria 1563157 Cupixi Virus 208899 endophyticamammarenavirus Pseudomonas Bacteria 312306 Curionopolis virus Virus490110 entomophila Pseudomonas Bacteria 2599595 Lymphocytic Virus 11623eucalypticola choriomeningitis mammarenavirus Pseudomonas BacteriaLyssavirus aravan Virus 211977 excibis Pseudomonas Bacteria 359110Lyssavirus Virus 90961 extremaustralis australis Pseudomonas Bacteria169669 Lyssavirus lagos Virus 38766 extremorientalis PseudomonasBacteria 2842355 Lyssavirus spp. Virus 11286 fakonensis PseudomonasBacteria 2841207 Lyssavirus bokeloh Virus 1072176 farris PseudomonasBacteria 2745492 Lyssavirus caucasicus Virus 249584 farsensisPseudomonas Bacteria 53410 Lyssavirus duvenhage Virus 38767 ficuserectaePseudomonas Bacteria 1674920 Lyssavirus irkut Virus 249583 fildesensisPseudomonas Bacteria 29435 Lyssavirus khujand Virus 237716 flavescensPseudomonas Bacteria 706570 Lyssavirus mokola Virus 12538 flexibilisPseudomonas Bacteria 1958950 Lyssavirus rabies Virus 11292 floridensisPseudomonas Bacteria 294 Lyssavirus shimoni Virus 746543 fluorescensPseudomonas Bacteria 1793966 Marisma mosquito Virus 1105173 fluvialisvirus Pseudomonas Bacteria 2762593 Marituba virus Virus 292278foliumensis Pseudomonas Bacteria 296 Marondera virus Virus 108092 fragiPseudomonas Bacteria 104087 Marrakai virus Virus 108088frederiksbergensis Pseudomonas Bacteria 200453 Massila virus Virusfulgida Pseudomonas Bacteria 47880 Matariya virus Virus 1272948 fulvaPseudomonas Bacteria 1149133 Matruh virus Virus 1678229 furukawaiiPseudomonas Bacteria 50340 Matucare virus Virus 908873 fuscovaginaePseudomonas Bacteria 1653853 Mayaro virus Virus 59301 gelidicolaPseudomonas Bacteria 78544 Mboke virus Virus 273342 gessardiiPseudomonas Bacteria 117681 Mburo virus Virus 2035534 gingeriPseudomonas Bacteria 1577705 Meaban virus Virus 35279 glareaePseudomonas Bacteria 1785145 Medjerda Valley Virus 1775957 glycinaevirus Pseudomonas Bacteria 2774461 Melao virus Virus 35515 gozinkensisPseudomonas Bacteria 158627 Meno virus Virus graminis PseudomonasBacteria 1421430 Mercadeo virus Virus 1708574 granadensis PseudomonasBacteria 1628277 Semliki Forest Virus 11033 gregormendelii virusPseudomonas Bacteria 129847 Sena Madureira Virus 1272957 grimontii virusPseudomonas Bacteria 1245526 Seoul virus Virus 1980490 guangdongensisPseudomonas Bacteria 1288410 Sepik virus Virus 44026 guariconensisPseudomonas Bacteria 310348 Serra Do Navio Virus 45768 guezennei virusPseudomonas Bacteria 1198456 Serra Norte virus Virus 1000649 guguanensisPseudomonas Bacteria 425504 Severe fever with Virus 1003835 guineaethrombocytopenia syndrome virus Pseudomonas Bacteria 2759165 Shamondavirus Virus 159150 guryensis Pseudomonas Bacteria 2600065 Shark Rivervirus Virus 2303490 haemolytica Pseudomonas Bacteria 53411 Shiant Islandvirus Virus halodenitrificans Pseudomonas Bacteria 28258 Shokwe virusVirus 273359 halodurans Pseudomonas Bacteria Shuni virus Virus 159148halosaccharolytica Pseudomonas Bacteria Silverwater virus Virus 1564099halosensibilis Pseudomonas Bacteria 2745504 Simbu Virus 35306hamedanensis orthobunyavirus Pseudomonas Bacteria 251654 Sin Nombrevirus Virus 1980491 helianthi Pseudomonas Bacteria 1608996 Sindbis virusVirus 11034 helleri Pseudomonas Bacteria 1471381 Sixgun City virus Virushelmanticensis Pseudomonas Bacteria 2213017 Skinner Tank virus Virus481886 huaxiensis Pseudomonas Bacteria 1247546 Snowshoe hare Virus 11580hunanensis virus Pseudomonas Bacteria 2707027 Sokoluk virus Virus 64317hutmensis Pseudomonas Bacteria 297 Soldado virus Virus 426791hydrogenothermophila Pseudomonas Bacteria 39439 Solwezi virus Virushydrogenovora Pseudomonas Bacteria 2493633 Somone virus Virushydrolytica Pseudomonas Bacteria 137658 Sororoca virus Virus 273354indica Pseudomonas Bacteria 404407 Souris virus Virus 2010246indoloxydans Pseudomonas Bacteria 2078786 South Bay virus Virus 1526514inefficax Pseudomonas Bacteria 2745503 South River virus Virus 45769iranensis Pseudomonas Bacteria 2710587 Spanish Culex Virus iridisflavivirus virus Pseudomonas Bacteria 2684212 Spanish Virus izuensisOchlerotatus flavivirus virus Pseudomonas Bacteria 256466 Spondwenivirus Virus 64318 japonica Pseudomonas Bacteria 77298 Sprivirus cyprinusVirus 696863 jessenii Pseudomonas Bacteria Sripur virus Virus 1620897jinanensis Pseudomonas Bacteria 198616 St. Abbs Head Virus jinjuensisvirus Pseudomonas Bacteria 2666183 St. Croix River Virus juntendi virusPseudomonas Bacteria 2293832 St. Louis Virus 11080 kairouanensisencephalitis virus Pseudomonas Bacteria 1055468 Stanfield virus Viruskarstica Pseudomonas Bacteria 2745482 Stratford virus Virus 44027kermanshahensis

TABLE 6 Exemplary list of viruses NCBI NCBI NCBI Taxonomy TaxonomyTaxonomy Name ID Name ID Name ID Aalivirus A 2169685 Enterovirus A138948 Pseudomonas 462590 virus Yua Aarhusvirus 2732762 Enterovirus B138949 Pseudoplusia dagda includens virus Aarhusvirus 2732763Enterovirus C 138950 Pseudotevenvirus 329381 katbat RB16 Aarhusvirus2732764 Enterovirus D 138951 Pseudotevenvirus 115991 luksen RB43Aarhusvirus 2732765 Enterovirus E 12064 Psimunavirus 2734265 mysterionpsiM2 Abaca bunchy 438782 Enterovirus F 1330520 Psipapillomavirus 11177762 top virus Abatino macacapox 2734574 Enterovirus G 106966Psipapillomavirus 2 2170170 virus Abbeymikolonvirus 2734213 EnterovirusH 310907 Psipapillomavirus 3 2170171 abbeymikolon Abouovirus 1984774Enterovirus I 2040663 Psittacid 50294 abouo alphaherpesvirus 1Abouovirus 1984775 Enterovirus J 1330521 Psittacine 2003673 daviesatadenovirus A Abutilon 1926117 Enterovirus K 2169884 Psittacine 2169709golden mosaic aviadenovirus B virus Abutilon 932071 Enterovirus L2169885 Psittacine 2734577 mosaic aviadenovirus C Bolivia virus Abutilon1046572 Entnonagintavirus 2734061 Psittacinepox 2169712 mosaic BrazilENT90 virus virus Abutilon 10815 Entoleuca 2734428 Pteridovirus 2734351mosaic virus entovirus filicis Abutilon 169102 Enytus Pteridovirus2734352 yellows virus montanus maydis ichnovirus Acadevirus 2733576Ephemerovirus 1972589 Pteropodid 2560693 PM116 adelaide alphaherpesvirus1 Acadevirus 2733577 Ephemerovirus 1972594 Pteropox virus 1873698 Pm5460berrimah Acadevirus 2733574 Ephemerovirus 1972593 Pteropus 1985395 PM85febris associated gemycircularvirus 1 Acadevirus 2733575 Ephemerovirus1972595 Pteropus 1985404 PM93 kimberley associated gemycircularvirus 10Acadianvirus 1982901 Ephemerovirus 1972596 Ptyasnivirus 1 2734501acadian koolpinyah Acadianvirus 1982902 Ephemerovirus 1972587Pukovnikvirus 540068 baee kotonkan pukovnik Acadianvirus 1982903Ephemerovirus 1972592 Pulverervirus 2170091 reprobate obodhiang PFR1Acanthamoeba 212035 Ephemerovirus 1972597 Puma lentivirus 12804polyphaga yata mimivirus Acanthocystis 322019 Epichloe 382962 Pumpkin2518373 turfacea festucae virus polerovirus chlorella virus 1 1 Acara2170053 Epinotia 166056 Pumpkin yellow 1410062 orthobunyavirus aporemamosaic virus granulovirus Achimota 2560259 Epiphyas 70600 Punavirus P110678 pararubulavirus 1 postvittana nucleopolyhed rovirus Achimota2560260 Epirus cherry 544686 Punavirus RCS47 2560452 pararubulavirus 2virus Achromobacter 2169962 Epizootic 100217 Punavirus SJ46 2560732virus Axp3 haematopoietic necrosis virus Acidianus 437444 Epizootic40054 Punique 2734468 bottle-shaped hemorrhagic phlebovirus virusdisease virus Acidianus 300186 Eponavirus 2734105 Punta Toro 1933186filamentous epona phlebovirus virus 2 Acidianus 346881 Epseptimavirus1982565 Puumala 1980486 filamentous 118970sal2 orthohantavirus virus 3Acidianus 346882 Epseptimavirus 491003 Pyrobaculum 1805492 filamentousEPS7 filamentous virus virus 6 1 Acidianus 346883 Epseptimavirus 2732021Pyrobaculum 270161 filamentous ev123 spherical virus virus 7 Acidianus346884 Epseptimavirus 2732022 Qadamvirus 2733953 filamentous ev329 SB28virus 8 Acidianus 512792 Epseptimavirus 2732023 Qalyub 1980527filamentous LVR16A orthonairovirus virus 9 Acidianus 309181Epseptimavirus 2732019 Qingdaovirus J21 2734135 rod-shaped mar003J3virus 1 Acidianus 693629 Epseptimavirus 2732024 Qingling 2560694spindle- S113 orthophasmavirus shaped virus 1 Acidianus 315953Epseptimavirus 2732025 Quail pea mosaic two-tailed S114 virus virusAcinetobacter 279006 Epseptimavirus 2732026 Quailpox virus 400570 virus133 S116 Acintetobacter Epseptimavirus 2732027 Quaranjavirus 688437virus B2 S124 johnstonense Acintetobacter Epseptimavirus 2732028Quaranjavirus 688436 virus B5 S126 quaranfilense Acionnavirus 2734078Epseptimavirus 2732029 Qubevirus durum 39803 monteraybay S132Acipenserid 2871198 Epseptimavirus 2732030 Qubevirus 39804 herpesvirus 2S133 faecium Aconitum 101764 Epseptimavirus 2732031 Quezon 2501382latent virus S147 mobatvirus Acrobasis Epseptimavirus 2732020Quhwahvirus 2283289 zelleri saus 132 kaihaidragon entomopoxvirusActinidia seed 2560282 Epseptimavirus 2732032 Quhwahvirus 2201441 bornelatent seafire ouhwah virus Actinidia 2024724 Epseptimavirus 2732033Quhwahvirus 2182400 virus 1 SH9 paschalis Actinidia 1112769Epseptimavirus 2732034 Rabbit associated 1985420 virus A STG2gemykroznavirus 1 Actinidia 1112770 Epseptimavirus 1540099 Rabbitfibroma 10271 virus B stitch virus Actinidia 1331744 Epseptimavirus2732035 Rabbit 11976 virus X Sw2 hemorrhagic disease virus Acute bee92444 Epsilonarterivirus 2501964 Rabovirus A 1603962 paralysis virushemcep Adana 2734433 Epsilonarterivirus 2501965 Rabovirus B 2560695phlebovirus safriver Adeno- 1511891 Epsilonarterivirus 2501966 RabovirusC 2560696 associated zamalb dependoparvo virus A Adeno- 1511892Epsilonpapillo 40537 Rabovirus D 2560697 associated mavirus 1dependoparvo virus B Adoxophyes 1993630 Epsilonpapillo 2169886Raccoonpox 10256 honmai mavirus 2 virus entomopoxvirus Adoxophyes 224399Epsilonpolyo 1891754 Radish leaf curl 435646 honmai mavirus bovis virusnucleopolyhed rovirus Adoxophyes 170617 Eptesipox 1329402 Radish mosaic328061 orana virus virus granulovirus Aedes aegypti Equid 10326 Radishyellow 319460 entomopoxvirus alphaherpesvirus 1 edge virus Aedes aegyptiEquid 80341 Rafivirus A Mosqcopia alphaherpesvirus virus 3 Aedes 341721Equid 10331 Rafivirus B 2560699 pseudoscutellaris alphaherpesvirusreovirus 4 Aegirvirus 2733888 Equid 39637 Rafivirus C SCBP42alphaherpesvirus 8 Aeonium 1962503 Equid 55744 Raleighvirus 2734266ringspot virus alphaherpesvirus 9 darolandstone Aeromonas Equid 12657Raleighvirus 2734267 virus 43 gammaherpes raleigh virus 2 Aeropyrum1157339 Equid 10371 Ramie mosaic 1874886 coil-shaped gammaherpes Yunnanvirus virus virus 5 Aeropyrum 700542 Equid 291612 Ranid 85655 pernixgammaherpes herpesvirus 1 bacilliform virus 7 virus 1 Aeropyrum 1032474Equine 1985379 Ranid 389214 pernix ovoid associated herpesvirus 2 virus1 gemycircularvirus 1 Aerosvirus 2733365 Equine 201490 Ranid 1987509 AS7encephalosis herpesvirus 3 virus Aerosvirus 2733364 Equine foamy 109270Ranunculus leaf 341110 av25AhydR2PP virus distortion virus Aerosvirus2733366 Equine 11665 Ranunculus mild 341111 ZPAH7 infectious mosaicvirus anemia virus Affertcholera 141904 Equine 129954 Ranunculus 341112mvirus mastadenovirus mosaic virus CTXphi A African 2560285 Equine129955 Raptor 691961 cassava mastadenovirus siadenovirus A mosaic BBurkina Faso virus African 10817 Equine 2723956 Raspberry bushy 12451cassava picobirnavirus dwarf virus mosaic virus African 2056161 Equinerhinitis 47000 Raspberry leaf 326941 eggplant A virus mottle virusmosaic virus African horse 40050 Equine 329862 Raspberry 12809 sicknessvirus torovirus ringspot virus African oil 185218 Eracentumvirus 1985737Rat associated 1985405 palm ringspot era103 gemycircularvirus virus 1African swine 10497 Eracentumvirus 2733579 Rat associated 2170126 fevervirus S2 porprismacovirus 1 Agaricus 2734345 Eragrostis 638358 Rattailcactus 1123754 bisporus curvula streak necrosis- alphaendornavirus 1virus associated virus Agaricus Eragrostis 1030595 Rattus norvegicus1679933 bisporus virus 4 minor streak polyomavirus 1 virus Agatevirus1910935 Eragrostis 496807 Rauchvirus BPP1 194699 agate streak virusAgatevirus 1910936 Erbovirus A 312185 Raven circovirus 345250 bobbAgatevirus 1910937 Erectites 390443 Ravinvirus N15 40631 Bp8pC yellowmosaic virus Ageratum 1260769 Eriborus Recovirus A 2560702 enationterebrans alphasatellite ichnovirus Ageratum 188333 Erinnyis ello 307444Red clover enation virus granulovirus associated luteovirus Ageratum1386090 Eriocheir 273810 Red clover 1323524 latent virus sinensiscryptic virus 2 reovirus Ageratum leaf 912035 Ermolevavirus 2733903 Redclover mottle 12262 curl Buea PGT2 virus betasatellite Ageratum leaf635076 Ermolevavirus 2733904 Red clover 12267 curl PhiKT necrotic mosaicCameroon virus betasatellite Ageratum leaf 2182585 Erskinevirus 2169882Red clover vein 590403 curl Sichuan asesino mosaic virus virus Ageratumleaf 333293 Erskinevirus 2169883 Red deerpox curl virus EaH2 virusAgeratum 169687 Erysimum 12152 Redspotted 43763 yellow leaf latent virusgrouper nervous curl necrosis virus betasatellite Ageratum 187850 Feline1987742 Reginaelenavirus 2734071 yellow vein associated rv3LV2017alphasatellite cyclovirus 1 Ageratum 185750 Feline 11978 Rehmannia425279 yellow vein calicivirus mosaic virus betasatellite Ageratum1454227 Feline foamy 53182 Rehmannia virus 1 2316740 yellow vein virusChina alphasatellite Ageratum 437063 Feline 11673 Reptilian 122203yellow vein immunodeficiency ferlavirus Hualian virus virus Ageratum1407058 Feline 11768 Reptilian 226613 yellow vein leukemia virusorthoreovirus India alphasatellite Ageratum 2010316 Feline 1170234Rerduovirus 1982376 yellow vein morbillivirus RER2 India betasatelliteAgeratum 915293 Felipivirus A Rerduovirus 1109716 yellow vein RGL3Singapore alphasatellite Ageratum 2010317 Felixounavirus 2560439Restivirus RSS1 2011075 yellow vein Alf5 Sri Lanka betasatelliteAgeratum 222079 Felixounavirus 1965378 Reston ebolavirus 186539 yellowvein AYO145A Sri Lanka virus Ageratum 44560 Felixounavirus 2560723Reticuloendotheliosis 11636 yellow vein BPS15Q2 virus virus Aghbyvirus2733367 Felsduovirus 2734062 Reyvirus rey 1983751 ISAO8 4LV2017Aglaonema 1512278 Felsduovirus 194701 Rhesus macaque 2170199 bacilliformFels2 simian foamy virus virus Agricanvirus 1984777 Felsduovirus 2734063Rhinolophus 2004965 deimos RE2010 associated gemykibivirus 1Agricanvirus 2560433 Felsduovirus 2734062 Rhinolophus 2004966 desertfox4LV2017 associated gemykibivirus 2 Agricanvirus 1984778 Felsduovirus194701 Rhinolophus bat 693998 Ea3570 Fels2 coronavirus HKU2 Agricanvirus1984779 Fernvirus 1921560 Rhinolophus 2501926 ray shelly ferrumequinumalphacoronavirus HuB-2013 Agricanvirus 1984780 Fernvirus 1921561Rhinovirus A 147711 simmy50 sitara Agricanvirus 1984781 Festuca leafRhinovirus B 147712 specialG streak cytorhabdovirus Agropyron 41763Fibralongavirus 2734233 Rhinovirus C 463676 mosaic virus fv2638A Agrotis208013 Fibralongavirus 2734234 Rhizidiomyces ipsilon QT1 virus multiplenucleopolyhed rovirus Agrotis 10464 Fibrovirus fs1 70203 Rhizoctonia1408133 segetum cerealis granulovirus alphaendornavirus 1 Agrotis1962501 Fibrovirus 1977140 Rhizoctonia 2560704 segetum VGJ magoulivirus1 nucleopolyhed rovirus A Agrotis 1580580 Ficleduovirus 2560473 Sabo2560716 segetum FCL2 orthobunyavirus nucleopolyhed rovirus B Agtrevirus1987994 Ficleduovirus 2560474 Saboya virus 64284 AG3 FCV1 Agtrevirus2169690 Fig badnavirus 1034096 Sacbrood virus 89463 SKML39 1 Aguacate2734434 Fig cryptic 882768 Saccharomyces 186772 phlebovirus virus 20SRNA narnavirus Ahlum Figulus Saccharum streak 683179 waterbornesublaevis virus virus entomopoxvirus Ahphunavirus 2733368 Figwort 10649Saclayvirus 2734138 Ahp1 mosaic virus Aci011 Ahphunavirus 2733369 Fijidisease 77698 Saclayvirus 2734139 CF7 virus Aci022 Ahtivirus 2734079Finch 400122 Saclayvirus 2734137 sagseatwo circovirus Aci05 Aichivirus A72149 Finkel-Biskis- 353765 Saetivirus fs2 1977306 Jinkins murinesarcoma virus Aichivirus B 194965 Finnlakevirus 2734591 Saetivirus VFJ1977307 FLIP Aichivirus C 1298633 Fionnbharthvirus 2955891 Saffronlatent 2070152 fionnbharth virus Aichivirus D 1897731 Fipivirus ASaguaro cactus 52274 virus Aichivirus E 1986958 Fipvunavirus 2560476Saguinine 2169901 Fpv4 gammaherpesvirus 1 Aichivirus F 1986959Firehammervirus 1190451 Saikungvirus 2169924 CP21 HK633 Ailurivirus A2560287 Firehammervirus 722417 Saikungvirus 2169925 CP220 HK75 Aino2560289 Firehammervirus 722418 Saimiri sciureus 1236410 orthobunyavirusCPt10 polyomavirus 1 Air potato 2560290 Fischettivirus 230871 Saimiriine10353 ampelovirus 1 C1 alphaherpesvirus 1 Akabane 1933178 Fishburnevirus1983737 Saimiriine 1535247 orthobunyavirus brusacoram betaherpesvirus 4Akhmeta virus 2200830 Flamingopox 503979 Saimiriine 10381 virusgammaherpesvirus 2 Alajuela 1933181 Flammulina 568090 Saint Florisorthobunyavirus velutipes phlebovirus browning virus Alasvirus 2501934Flaumdravirus 2560665 Saint Louis 11080 muscae KIL2 encephalitis virusAlcelaphine 35252 Flaumdravirus 2560666 Saint Valerien gammaherpes KIL4virus virus 1 Alcelaphine 138184 Fletchervirus 1980966 Sakhalin 1980528gammaherpes CP30A orthonairovirus virus 2 Alcube 2734435 Gaiavirus gaia1982148 Sakobuvirus A 1659771 phlebovirus Alcyoneusvirus 2560541Gaillardia 1468172 Sal Vieja virus 64301 K641 latent virusAlcyoneusvirus 2560545 Gairo 1535802 Salacisavirus 2734140 RaK2mammarenavirus pssm2 Alefpapilloma 2169692 Gajwadongvirus 2733916Salanga 2734471 virus 1 ECBP5 phlebovirus Alenquer 2734436Gajwadongvirus 2733917 Salasvirus phi29 10756 phlebovirus PP99Alexandravirus 2734080 Galaxyvirus 2560298 Salchichonvirus 298338 AD1abidatro LP65 Alexandravirus 2734081 Galaxyvirus 2560303 Salehabad1933188 alexandra galaxy phlebovirus Alfalfa Galinsoga 60714 Salemsalemvirus 2560718 betanucleorha mosaic virus bdovirus Alfalfa crypticGallid 10386 Salivirus A 1330524 virus 1 alphaherpesvirus 1 Alfalfa1770265 Gamaleyavirus 1920761 Salmo 2749930 enamovirus 1 Sb1aquaparamyxovirus Alfalfa leaf 1306546 Gambievirus 2501933 Salmongillpox 2734576 curl virus bolahunense virus Alfalfa mosaic 12321 Gamboa1933270 Saphexavirus 1982380 virus orthobunyavirus VD13 Alfalfa virus S1985968 Gammaarterivirus 2499678 Sapporo virus 95342 lacdeh Algerian515575 Gammanucleor-habdovirus 2748968 Sarcochilus virus 104393watermelon maydis Y mosaic virus Allamanda 452758 Gammapapillomavirus333926 Sashavirus sasha 2734275 leaf curl virus 1 Allamanda 1317107Gammapapillomavirus 1175852 Sasquatchvirus 2734143 leaf mottle 10 Y3distortion virus Alligatorweed Gammapapillomavirus 1513256 SasvirusBFK20 2560392 stunting virus 11 Allium cepa 2058778 Gayfeather 578305Satsuma dwarf 47416 amalgavirus 1 mild mottle virus virus Allium cepa2058779 Gecko 2560481 Sauletekiovirus 2734030 amalgavirus 2reptillovirus AAS23 Allium virus 317027 Gelderlandvirus 2560727 SaumarezReef 40012 X melville virus Allpahuayo 144752 Gelderlandvirus 1913658Saundersvirus 2170234 mammarenavirus s16 Tp84 Almendravirus 1972686Gelderlandvirus 1913657 Sauropus leaf 1130981 almendras stml198 curlvirus Almendravirus 1972683 Gelderlandvirus 2560734 Sawgrhavirus 2734397arboretum stp4a connecticut Almendravirus 1972684 Gentian 182452Sawgrhavirus 2734398 balsa mosaic virus longisland Almendravirus 1972687Gentian ovary 1920772 Sawgrhavirus 2734399 chico ringspot virus mintoAlmendravirus 1972685 Geotrupes Sawgrhavirus 2734400 cootbay sylvaticussawgrass entomopoxvirus Almendravirus 2734366 Gequatrovirus 1986034Scale drop 1697349 menghai G4 disease virus Bat associated 1987731Gequatrovirus 1910968 Scallion mosaic 157018 cyclovirus 6 ID52 virus Batassociated 1987732 Gequatrovirus 1910969 Scapularis 2734431 cyclovirus 7talmos ixovirus Bat associated 1987733 Gerygone 1985381 Scapunavirus2560792 cyclovirus 8 associated scap1 gemycircularvirus 1 Bat associated1987734 Gerygone 1985382 Scheffersomyces 1300323 cyclovirus 9 associatedsegobiensis virus gemycircularvirus 2 L Bat 1913643 Harrisina 115813Schefflera 2169729 coronavirus brillians ringspot virus CDPHE15granulovirus Bat 1244203 Harrisonvirus 1982221 Schiekvirus 2560422coronavirus harrison EFDG1 HKU10 Bat Hp- 2501961 Harvey 11807Schiekvirus 2734044 betacoronavirus murine EFP01 Zhejiang2013 sarcomavirus Bat 1146877 Hautrevirus 1982895 Schiekvirus 2734045 mastadenovirusA hau3 EfV12 Bat 1146874 Havel River 254711 Schistocerca mastadenovirusB virus gregaria entomopoxvirus Bat 2015370 Hawkeyevirus 2169910Saphexavirus 1982380 mastadenovirus C hawkeye VD13 Bat 2015372 Hazara1980522 Sophora yellow 2169837 mastadenovirus D orthonairovirus stuntalphasatellite 5 Bat 2015374 Heartland 2747342 Sorex araneus 2734504mastadenovirus E bandavirus coronavirus T14 Bat 2015375 Hebius Sorexaraneus 2560769 mastadenovirus F tobanivirus 1 polyomavirus 1 Bat2015376 Hedgehog 1965093 Sorex coronatus 2560770 mastadenovirus Gcoronavirus 1 polyomavirus 1 Bat Hedwigvirus 2560502 Sorex minutus2560771 mastadenovirus H hedwig polyomavirus 1 Bat Hedyotis 1428190Sorghum 107804 mastadenovirus I uncinella chlorotic spot yellow mosaicvirus virus Bat Hedyotis 1428189 Sorghum mosaic 32619 mastadenovirus Jyellow mosaic virus betasatellite Batai 2560341 Heilongjiangvirus2734110 Sororoca 2560772 orthobunyavirus Lb orthobunyavirus Batama1933177 Helenium 12171 Sortsnevirus 2734190 orthobunyavirus virus SIME279 Batfish 2560342 Helianthus 2184469 Sortsnevirus 2734189actinovirus annuus sortsne alphaendornavirus Bavaria virus 2560343Helicobasidium 675833 Sosuga 2560773 mompa pararubulavirusalphaendornavirus 1 Baxtervirus 2169730 Helicobasidium 344866 Soupsvirussoups 1982563 baxterfox mompa partitivirus V70 Baxtervirus 2169731Helicobasidium 196690 Soupsvirus 2560510 yeezy mompa strosahl totivirus1-17 Baylorvirus 2734055 Helicoverpa 489830 Soupsvirus wait 2560513bv1127AP1 armigera granulovirus Baylorvirus 376820 Helicoverpa 51313Souris 2169997 PHL101 armigera mammarenavirus nucleopolyhed rovirusBayou 1980459 Helicoverpa 37206 Sourvirus sour 2560509 orthohantavirusarmigera stunt virus Bcepfunavirus 417280 Heliothis 10290 South African63723 bcepF1 armigera cassava mosaic entomopoxvirus virus Bcepmuvirus264729 Heliothis 113366 Southern bean 12139 bcepMu virescens mosaicvirus ascovirus 3a Bcepmuvirus 431894 Heliothis zea 29250 Southerncowpea 196398 E255 nudivirus mosaic virus Bdellomicrovirus 1986027Helleborus 592207 Southern 1159195 MH2K mosaic virus elephant seal virusBdellovibrio Helleborus net 592206 Southern rice 519497 virus MAC1necrosis virus black-streaked dwarf virus Beak and 77856Helminthosporium 2560520 Southern tomato 591166 feather diseasevictoriae virus virus virus 145S Bean calico 31602 Helminthosporium45237 Sowbane mosaic 378833 mosaic virus victoriae virus virus 190S Beanchlorosis 1227354 Helsettvirus 2733626 Soybean 1985413 virus fPS53associated gemycircularvirus 1 Bean common 43240 Helsettvirus 2733628Sophora yellow 2169837 mosaic fPS54ocr stunt necrosis virusalphasatellite 5 Bean common 12196 Helsettvirus 2733627 Sorex araneus2734504 mosaic virus fPS59 coronavirus T14 Bean dwarf 10838 Helsettvirus2733625 Sorex araneus 2560769 mosaic virus fPS9 polyomavirus 1 Beangolden 10839 Helsingorvirus 1918193 Sorex coronatus 2560770 mosaic virusCba121 polyomavirus 1 Bean golden 220340 Helsingorvirus 1918194 Sorexminutus 2560771 yellow mosaic Cba171 polyomavirus 1 virus Bean leaf2004460 Jujube 2020956 Sorghum 107804 crumple virus mosaic- chloroticspot associated virus virus Bean leafroll 12041 Jun 2560536 Sorghummosaic 32619 virus jeilongvirus virus Bean mild Juncopox Sororoca2560772 mosaic virus virus orthobunyavirus Bean necrotic 2560344 Jutiapavirus 64299 Sortsnevirus 2734190 mosaic IME279 orthotospovirus Bean pod12260 Jwalphavirus 2169963 Switchgrass 2049938 mottle virus jwalphamosaic- associated virus Bean rugose 128790 Kabuto 2747382 Symapivirus Amosaic virus mountain uukuvirus Bean white 2169732 Kadam virus 64310Synechococcus 2734100 chlorosis virus SRIM12-08 mosaic virus Bean yellow267970 Kadipiro virus 104580 Synedrella leaf 1544378 disorder virus curlalphasatellite Bean yellow 714310 Kaeng Khoi 1933275 Synedrella 1914900mosaic orthobunyavirus yellow vein Mexico virus clearing virus Beanyellow 12197 Kafavirus 2733923 Synetaeris mosaic virus SWcelC56tenuifemur ichnovirus Bear Canyon 192848 Kafunavirus 1982588 Syngnathid2734305 mammarenavirus KF1 ichthamaparvovirus 1 Beauveria 1740646Kagunavirus 2560464 Synodus 2749934 bassiana golestan synodonviruspolymycovirus 1 Beauveria 1685109 Kagunavirus 1911008 Tabernariusvirus2560691 bassiana K1G tabernarius victorivirus 1 Bebaru virus 59305Kagunavirus 1911010 Tacaiuma 611707 K1H orthobunyavirus Beecentumtre10778 Kagunavirus 1911007 Tacaribe 11631 virus B103 K1ind1mammarenavirus Beet black 196375 Kagunavirus 1911009 Tacheng 2734606scorch virus K1ind2 uukuvirus Beet chlorosis 131082 Kagunavirus 2734197Tahyna 2560796 virus RP180 orthobunyavirus Beet cryptic 509923 Merremia77813 Tangaroavirus 2733962 virus 1 mosaic virus tv951510a Beet cryptic912029 Mesta yellow 1705093 Tankvirus tank 1982567 virus 2 vein mosaicalphasatellite Beet cryptic 29257 Mesta yellow 508748 Tapara 2734474virus 3 vein mosaic phlebovirus Bahraich virus Beet curly top 391228Metamorphoo 2734253 Tapirape 2560798 Iran virus virus fireman pacuvirusBeet curly top 10840 Metamorphoo 2734254 Tapwovirus cesti 2509383 virusvirus metamorphoo Beet mild 156690 Metamorphoo 2734255 Taranisvirus2734146 yellowing virus robsfeet taranis virus Beet mosaic 114921Metrivirus 2560269 Taro bacilliform 1634914 virus ME3 CH virus Beetnecrotic 31721 Mguuvirus 2733593 Taro bacilliform 178354 yellow veinJG068 virus virus Beet 72750 Microbacterium Tarumizu 2734340pseudoyellows virus coltivirus virus MuffinTheCat [2] Beet ringspot191547 Microcystis 340435 Tataguine 2560799 virus virus Ma-orthobunyavirus LMM01 Beet soil- 76343 Microhyla Taterapox virus 28871borne mosaic letovirus 1 virus Beet soil- 46436 Micromonas 338781Taupapillomavirus 1176148 borne virus pusilla 1 reovirus Beet virus Q71972 Micromonas 373996 Taupapillomavirus 1513274 pusilla virus 2 SP1Beet western 12042 Microplitis Taupapillomavirus 1961786 yellows viruscroceipes 3 bracovirus Beet yellow 35290 Microtus 2006148Taupapillomavirus 2170222 stunt virus arvalis 4 polyomavirus 1 Beetyellows 12161 Mukerjeevirus 2734186 Taura syndrome 142102 virus mv52B1virus Beetle mivirus Mulberry 1227557 Tawavirus JSF7 2733965 badnavirus1 Beetrevirus 2560656 Mulberry 1631303 Tea plant 2419939 B3 mosaic dwarfnecrotic ring associated blotch virus virus Beetrevirus 2560663 Mulberry1527441 Tefnutvirus 2734147 JBD67 mosaic leaf siom18 roll associatedvirus Beetrevirus 2560664 Mulberry Tegunavirus r1rt 1921705 JD18ringspot virus Beetrevirus 2560675 Mulberry vein Tegunavirus 1921706PM105 banding yenmtg1 associated orthotospovirus Beihai Mule deerpox304399 Tehran 2734475 picobirnavirus virus phlebovirus Beilong 2560345Mume virus A 2137858 Telfairia golden 2169737 jeilongvirus mosaic virusBell pepper 354328 Mumps 2560602 Telfairia mosaic 1859135alphaendornavirus orthorubulavirus virus Bell pepper 368735 Mungbean2010322 Tellina virus 359995 mottle virus yellow mosaic betasatelliteBelladonna 12149 Mukerjeevirus 2734186 Tellina virus 1 321302 mottlevirus mv52B1 Bellamyvirus 2734095 Mulberry 1227557 Telosma mosaic 400394bellamy badnavirus 1 virus Bellavista 2560346 Mulberry 1631303 Tembusuvirus 64293 orthobunyavirus mosaic dwarf associated virus Bellflower1720595 Mycobacterium 1993864 Tensaw 2560800 vein chlorosis virusorthobunyavirus virus Tweety Bellflower 1982660 Mycobacterium 1993860Tent-making bat 1508712 veinal mottle virus Wee hepatitis B virus virusBeluga whale 694015 Mycobacterium 1993859 Teseptimavirus 2733885coronavirus virus YpsPG SW1 Wildcat Bendigovirus 2560495 Mycoreovirus311228 Testudine GMA6 1 orthoreovirus Benedictvirus 1071502 Mycoreovirus404237 Testudinid 2560801 cuco 2 alphaherpesvirus 3 Benedictvirus1993876 Mycoreovirus 311229 Tete 35319 tiger 3 orthobunyavirus Benevides2170054 Mylasvirus 1914020 Tetterwort vein 1712389 orthobunyaviruspersius chlorosis virus Bequatrovirus 1984785 Mynahpox 2169711 Teviot2560803 avesobmore virus pararubulavirus Bequatrovirus 1918005 MyodesThailand 1980492 B4 coronavirus orthohantavirus 2JL14 Bequatrovirus1918006 Myodes 2006147 Thalassavirus 2060093 bigbertha glareolusthalassa polyomavirus 1 Bequatrovirus 1918007 Myodes 2560609Thaumasvirus 2734148 riley jeilongvirus stim4 Bequatrovirus 1918008Myodes 2560610 Thermoproteus 292639 spock narmovirus tenax sphericalvirus 1 Bequatrovirus 1918009 Myohalovirus 1980944 Thermoproteus 10479troll phiH tenax virus 1 Berhavirus 2509379 Noxifervirus 2560671 Thermusvirus 1714273 beihaiense noxifer IN93 Berhavirus 2509380 Ntaya virus64292 Thermus virus 1714272 radialis P23-77 Berhavirus 2509381 Ntepes2734464 Thetaarterivirus 2501999 sipunculi phlebovirus kafubaBerisnavirus 1 2734518 Nuarterivirus Thetaarterivirus 2502000 guemelmikelba 1 Cacao yellow 12150 Nudaurelia 85652 Thetapapillomavirus 197772mosaic virus capensis beta 1 virus Cacao yellow 2169726 Nudaurelia 12541Thetapolyomavirus 1891755 vein banding capensis censtriata virus omegavirus Cache Valley 2560364 Nupapillomavirus 334205 Thetapolyomavirus2218588 orthobunyavirus 1 trebernacchii Cachoeira 2560365 Nyando 1933306Thetapolyomavirus 2170103 Porteira orthobunyavirus trepennelliiorthobunyavirus Cacipacore 64305 Nyavirus 644609 Thetisvirus ssm12734149 virus midwayense Cactus mild 229030 Nyavirus 644610 Thiafora1980529 mottle virus nyamaniniense orthonairovirus Cactus virus 2Nyavirus 1985708 Thimiri 1819305 sierranevadaense orthobunyavirus Cactusvirus X 112227 Nyceiraevirus 2560506 Thin paspalum 1352511 nyceiraeasymptomatic virus Cadicivirus A 1330068 Nyctalus 2501928 Thistle mottlevelutinus virus alphacoronavirus SC-2013 Cadicivirus B 2560366Nylanderia 1871153 Thogotovirus 11318 fulva virus 1 dhorienseCaenorhabditi Nymphadoravirus 2170041 Thogotovirus 11569 elegans Cer1kita thogotoense virus Caenorhabditi Nymphadoravirus 2560507 Thomixvirus2560804 elegans nymphadora OH3 Cer13 virus Caeruleovirus 1985175Nymphadoravirus 2170042 Thornevirus 2560336 Bc431 zirinka SP15Caeruleovirus 1985176 Oat blue 56879 Thosea asigna 83810 Bcp1 dwarfvirus virus Caeruleovirus 1985177 Oat chlorotic 146762 Thottopalayam2501370 BCP82 stunt virus thottimvirus Caeruleovirus 1985178 Oat dwarf497863 Thunberg 299200 BM15 virus fritillary mosaic virus Caeruleovirus1985179 Oat golden 45103 Thysanoplusia 101850 deepblue stripe virusorichalcea nucleopolyhedro virus Caeruleovirus 1985180 Oxbow 1980484Tiamatvirus 268748 JBP901 orthohantavirus PSSP7 Cafeteria 1513235Oxyplax 2083176 Tibetan frog 2169919 roenbergensis ochracea hepatitis Bvirus virus nucleopolyhed rovirus Cafeteriavirus- 1932923 Paadamvirus2733939 Tibrovirus 1987018 dependent RHEph01 alphaekpoma mavirus Caimito2734421 Pacific coast Tibrovirus 2170224 pacuvirus uukuvirus beatriceCajanus cajan Pacui 2560617 Tibrovirus 1987019 Panzee virus pacuvirusbetaekpoma Caladenia 1198147 Paenibacillus Tibrovirus 1972586 virus Avirus Willow coastal Calanthe mild 73840 Pagavirus 2733940 Tibroviruscongo 1987017 mosaic virus S05C849 Cali 2169993 Pagevirus 1921185Tibrovirus 1987013 mammarenavirus page sweetwater Calibrachoa 204928Pagevirus 1921186 Tibrovirus 1972584 mottle virus palmer tibrogarganCalifornia 1933264 Pagevirus 1921187 Tick associated 2560805encephalitis pascal circovirus 1 orthobunyavirus California 2170175Pagevirus 1921188 Tick associated 2560806 reptarenavirus pony circovirus2 Caligid Pagevirus 1921189 Tick-borne 11084 hexartovirus pookieencephalitis virus Caligrhavirus 2560367 Pagoda yellow 1505530 Ticophebovirus 2734476 caligus mosaic associated virus Caligrhavirus 2560551Paguronivirus 2508237 Tidunavirus 2560834 lepeophtheirus 1 pTD1Caligrhavirus 2560736 Pahexavirus 1982252 Tidunavirus 2560833salmonlouse ATCC29399BC VP4B Calla lily 2560368 Pahexavirus 1982303Tiger puffer 43764 chlorotic spot pirate nervous necrosisorthotospovirus virus Calla lily 243560 Pahexavirus 1982304 Tigray2560807 latent virus procrass 1 orthohantavirus Callistephus 1886606Pahexavirus 1982305 Tigrvirus E122 431892 mottle virus SKKYCallitrichine 106331 Pahexavirus 1982306 Tigrvirus E202 431893gammaherpes solid virus 3 Calopogonium Pahexavirus 1982307 Tobacco leafcurl 439423 yellow vein stormborn Comoros virus virus Camel 2169876Pahexavirus 1982308 Tobacco leaf curl 336987 associated wizzo Cuba virusdrosmacovirus 1 Camel 2169877 Pahsextavirus 2733975 Tobacco leaf curl2528965 associated pAh6C Dominican drosmacovirus 2 Republic virus Camel2170105 Pairvirus 2733941 Tobacco leaf curl 2010326 associated Lo5R7ANSJapan porprismacovirus 1 betasatellite Camel 2170106 Pakpunavirus1921409 Tobacco leaf curl 2010327 associated CAb02 Patnaporprismacovirus 2 betasatellite Camel 2170107 Pahexavirus 1982303Tobacco leaf curl 905054 associated pirate Pusa virus porprismacovirus 3Camel 2170108 Pahexavirus 1982304 Tobacco leaf curl 409287 associatedprocrass 1 Thailand virus porprismacovirus 4 Camelpox 28873 Pahexavirus1982305 Tobacco leaf curl 211866 virus SKKY Yunnan virus Campana 2734442Pea necrotic 753670 Tobacco leaf curl 223337 phlebovirus yellow dwarfZimbabwe virus virus Campoletis Pea seed- 12208 Tobacco leaf 196691aprilis borne mosaic rugose virus ichnovirus virus Campoletis Pea stem199361 Veracruzvirus 1032892 flavicincta necrosis virus heldanichnovirus Camptochironomus Pea streak 157777 Veracruzvirus 2003502tentans virus rockstar entomopoxvirus Campylobacter 1006972 Pea yellow1436892 Verbena latent 134374 virus IBB35 stunt virus virus Camvirus1982882 Peach 471498 Verbena virus Y 515446 amela chlorotic mottle virusCamvirus 1982883 Peach latent 12894 Vernonia crinkle 1925153 CAM mosaicviroid virus Canary 142661 Peach 2169999 Vernonia yellow 666635circovirus marafivirus D vein betasatellite Canarypox 44088 Peach mosaic183585 Vernonia yellow 2169908 virus virus vein Fujian alphasatelliteCandida Peach rosette 65068 Vernonia yellow 2050589 albicans Tca2 mosaicvirus vein Fujian virus betasatellite Candida Peanut 35593 Vernoniayellow 1001341 albicans Tca5 chlorotic vein Fujian virus virus streakvirus Candiru 1933182 Peanut clump 28355 Vernonia yellow 367061phlebovirus virus vein virus Canid 170325 Peanut yellow Versovirus2011076 alphaherpesvirus 1 mosaic virus VfO3K6 Canine 1985425 Pearblister 12783 Verticillium 759389 associated canker viroid dahliaegemygorvirus 1 chrysovirus 1 Canine 1194757 Peaton 2560627 Vesicular35612 circovirus orthobunyavirus exanthema of swine virus Canine 10537Peatvirus 2560629 Vesiculovirus 1972579 mastadenovirus A peat2 alagoasCanine 11232 Pecan mosaic- 1856031 Vesiculovirus 1972567 morbillivirusassociated bogdanovac virus Canna yellow 2560371 Pecentumvirus 40523Whitefly- 2169744 mottle A511 associated associated begomovirus 7 virusCanna yellow 419782 Penicillum 2734569 White-tufted-ear 2170205 mottlevirus brevicompactum marmoset simian polymycovirus 1 foamy virus Cannayellow 433462 Pennisetum 221262 Whitewater 46919 streak virus mosaicvirus Arroyo mammarenavirus Cannabis 1115692 Pepino mosaic Wifcevirus2734154 cryptic virus virus [3] ECML117 Cano 1980463 Pepo aphid- 1462681Wifcevirus 2734155 Delgadito borne yellows FEC19 orthohantavirus virusCanoevirus 2734056 Pepper chat 574040 Wifcevirus WFC 2734156 canoe fruitviroid Cao Bang 1980464 Pepper 2734493 Wifcevirus WFH 2734157orthohantavirus chlorotic spot orthotospovirus Caper latent 1031708Phietavirus X2 320850 Wigeon 1159908 virus coronavirus HKU20 Capim1933265 Phifelvirus 1633149 Wild cucumber 70824 orthobunyavirus FL1mosaic virus Capistrivirus 2011077 Phikmvvirus 2733349 Wild melon KSF115pyo banding virus Capraria 2049955 Phlox virus S 436066 Wild onion1862127 yellow spot symptomless virus virus Caprine 39944 Phnom Penh64894 Wild potato 187977 alphaherpesvirus 1 bat virus mosaic virusCaprine 11660 Phocid 47418 Wild tomato 400396 arthritis alphaherpesvirusmosaic virus encephalitis 1 virus Caprine 135102 Phocid 47419 Wild Vitislatent 2560839 gammaherpes gammaherpes virus virus 2 virus 2 Caprine2560372 Phocid 2560643 Wilnyevirus 2560486 respirovirus 3 gammaherpesbillnye virus 3 Capsicum 2560373 Phocine 11240 Wilsonroadvirus 2734007chlorosis morbillivirus Sd1 orthotospovirus Capsicum 2734586 PholetesorWinged bean 2169693 India ornigis alphaendornavirus alphasatellitebracovirus 1 Captovirus 235266 Phthorimaea 192584 Winklervirus 2560752AFV1 operculella chi14 granulovirus Capuchin 2163996 Phutvirus 2733655Wiseana signata 65124 monkey PPpW4 nucleopolyhedro hepatitis B virusvirus Caraparu 1933290 Phyllosphere Wissadula golden 51673orthobunyavirus sclerotimonavirus mosaic virus Carbovirus 2136037Physalis 72539 Wissadula yellow 1904884 queenslandense mottle virusmosaic virus Dyonupapillo 1513250 Physarum Wisteria 1973265 mavirus 1polycephalum badnavirus 1 Tp1 virus Dyoomegapap 1918731 Phytophthora310750 Wisteria vein 201862 illomavirus 1 alphaendornavirus 1 mosaicvirus Dyoomikronp 1513251 Picardvirus 2734264 Witwatersrand 2560841apillomavirus 1 picard orthobunyavirus Dyophipapillo 1920493 Pidgey2509390 Wizardvirus 2170253 mavirus 1 pidchovirus twister6 Dyopipapillo1513252 Piedvirus 2733947 Wizardvirus 2170254 mavirus 1 IMEDE1 wizardDyopsipapillo 1920498 Pienvirus 2733373 Woesvirus woes 1982751 mavirus 1R801 Dyorhopapillo 1513253 Pifdecavirus 2733657 Wolkberg 2170059 mavirus1 IBBPF7A orthobunyavirus Dyosigmapapi 1513254 Plum bark 675077 Wongorrvirus 47465 llomavirus 1 necrosis stem pitting- associated virusDyotaupapillo 1932910 Plum pox 12211 Wongtaivirus 2169922 mavirus 1virus HK542 Dyothetapapill 1235662 Plumeria 1501716 Woodchuck 35269omavirus 1 mosaic virus hepatitis virus Dyoupsilonpa 1932912 Plutella98383 Woodruffvirus 1982746 pillomavirus 1 xylostella TP1604granulovirus Dyoxipapillo 1513255 Poa semilatent 12328 Woodruffvirus1982747 mavirus 1 virus YDN12 Dyoxipapillo 2169881 Poaceae 1985392Woolly monkey 68416 mavirus 2 associated hepatitis B virusgemycircularvirus 1 Dyozetapapill 1177766 Podivirus 2733948 Woollymonkey 11970 omavirus 1 S05C243 sarcoma virus Eapunavirus 2733615Poecivirus A 2560644 Wound tumor 10987 Eap1 virus East African 223262Pogseptimavirus 2733996 Wphvirus 2560329 cassava PG07 BPS10C mosaicCameroon virus East African 393599 Pogseptimavirus 2733997 WphvirusBPS13 1987727 cassava VspSw1 mosaic Kenya virus East African 223264Poindextervirus 2734196 Wphvirus hakuna 1987729 cassava BL10 mosaicMalawi virus East African 62079 Poindextervirus 2748760 Wphvirus 1987728cassava rogue megatron mosaic virus East African 223275 Poinsettia305785 Wphvirus WPh 1922328 cassava latent virus mosaic Zanzibar virusEast Asian 2734556 Poinsettia 113553 Wuchang 1980542 Passiflora mosaicvirus cockroach distortion orthophasmavirus virus 1 East Asian 341167Pokeweed 1220025 Wuhan mivirus 2507319 Passiflora mosaic virus virusEastern 2170195 Pokrovskaiavirus 2733374 Wuhan mosquito 1980543chimpanzee orthophasmavirus simian foamy fHe Yen301 1 virus Easternequine 11021 Pokrovskaiavirus 2733375 Wuhan mosquito 1980544encephalitis pv8018 orthophasmavirus virus 2 Eastern 2734571 Polar bearWuhanvirus 2733969 kangaroopox mastadenovirus PHB01 virus AEastlansingvirus 2734004 Pollockvirus 2170215 Wuhanvirus 2733970 Sf12pollock PHB02 Echarate 2734447 Pollyceevirus 2560679 Wumivirus 2509286phlebovirus pollyC millepedae Echinochloa 42630 Polybotosvirus 2560286Wumpquatrovirus 400567 hoja blanca Atuph07 WMP4 tenuivirus EchinochloaPolygonum 430606 Wumptrevirus 440250 ragged stunt ringspot WMP3 virusorthotospovirus Eclipta yellow 2030126 Pomona bat 2049933 Wutai mosquito1980612 vein hepatitis B phasivirus alphasatellite virus Eclipta yellow875324 Pongine 159603 Wyeomyia 273350 vein virus gammaherpesorthobunyavirus virus 2 Eclunavirus 2560414 Poplar mosaic 12166Xanthophyllomyces 1167690 EcL1 virus dendrorhous virus L1A Ectocarpus2083183 Popoffvirus 2560283 Xanthophyllomyces 1167691 fasciculatus pv56dendrorhous virus a virus L1B Ectocarpus 37665 Porcine 1985393 Xapuri2734417 siliculosus associated mammarenavirus virus 1 gemycircularvirus1 Ectocarpus Potato virus Y 12216 Xestia c-nigrum 51677 siliculosusgranulovirus virus a Ectromelia 12643 Potato yellow 2230887 Xiamenvirus1982373 virus blotch virus RDJL1 Ectropis 59376 Potato yellow 223307Xiamenvirus 1982374 obliqua mosaic RDJL2 nucleopolyhedrovirus Panamavirus Ectropis 1225732 Potato yellow 10827 Xilang striavirus 2560844obliqua virus mosaic virus Edenvirus 2734230 Potato yellow 103881Xinzhou mivirus 2507320 eden vein virus Edge Hill 64296 Pothos latent44562 Xipapillomavirus 10561 virus virus 1 Efquatrovirus 2560415 Potosi2560646 Xipapillomavirus 1513273 AL2 orthobunyavirus 2 Efquatrovirus2560416 Poushouvirus 2560396 Yokohamavirus 1980942 AL3 Poushou PEi21Efquatrovirus 2560417 Pouzolzia 1225069 Yokose virus 64294 AUEF3 goldenmosaic virus Efquatrovirus 2560424 Primate T- 194443 Yoloswagvirus2734158 EcZZ2 lymphotropic yoloswag virus 3 Efquatrovirus 2560420Primolicivirus 2011081 Yongjia 2734607 EF3 Pf1 uukuvirus Efquatrovirus2560421 Primula 1511840 Youcai mosaic 228578 EF4 malacoides virus virus1 Efquatrovirus 2560425 Priunavirus 2560652 Yunnan orbivirus 306276EfaCPT1 PR1 Efquatrovirus 2560426 Privet ringspot 2169960 Yushanvirus2733978 IME196 virus Spp001 Efquatrovirus 2560427 ProchlorococcusYushanvirus 2733979 LY0322 virus PHM1 SppYZU05 Efquatrovirus 2560428Prospect Hill 1980485 Yuyuevirus 2508254 PMBT2 orthohantavirusbeihaiense Efquatrovirus 2560429 Protapanteles Yuyuevirus 2508255SANTOR1 paleacritae shaheense bracovirus Efquatrovirus 2560430Providence 213633 Zaire ebolavirus 186538 SHEF2 virus Efquatrovirus2560431 Prune dwarf 33760 Zaliv Terpeniya 2734608 SHEF4 virus uukuvirusEfquatrovirus 2560432 Prunus latent 2560653 Zantedeschia 270478 SHEF5virus mild mosaic virus Eganvirus EtG 2734059 Prunus 37733 Zarhavirus2734410 necrotic zahedan ringspot virus Eganvirus 29252 Przondovirus2733672 Zika virus 64320 ev186 KN31

The cascade assays described herein are particularly well-suited forsimultaneous testing of multiple targets. Pools of two to 10,000 targetnucleic acids of interest may be employed, e.g., pools of 2-1000, 2-100,2-50, or 2-10 target nucleic acids of interest. Further testing may beused to identify the specific member of the pool, if warranted.

While the methods described herein do not require the target nucleicacid of interest to be DNA (and in fact it is specifically contemplatedthat the target nucleic acid of interest may be RNA), it is understoodby those in the field that a reverse transcription step to converttarget RNA to cDNA may be performed prior to or while contacting thebiological sample with the composition.

Nucleic Acid-Guided Nucleases

The cascade assays comprise nucleic acid-guided nucleases in thereaction mix, either provided as a protein, a coding sequence for theprotein, or, in many embodiments, in a ribonucleoprotein (RNP) complex.In some embodiments, the one or more nucleic acid-guided nucleases inthe reaction mix may be, for example, a Cas nucleic acid-guidednuclease. Any nucleic acid-guided nuclease having both cis- andtrans-cleavage activity may be employed, and the same nucleicacid-guided nuclease may be used for both RNP complexes or differentnucleic acid-guided nucleases may be used in RNP1 and RNP2. For example,RNP1 and RNP2 may both comprise Cas12a nucleic acid-guided nucleases, orRNP1 may comprise a Cas13 nucleic acid-guided nuclease and RNP2 maycomprise a Cas12a nucleic acid-guided nuclease or vice versa. Inembodiments where a variant nucleic acid-guided nuclease is employed,only RNP2 will comprise the variant, and RNP1 may comprise either aCas12a or Cas13 nucleic acid-guided nuclease. In embodiments where avariant nucleic acid-guided nuclease is not employed, either or bothRNP1 and RNP2 can comprise a Cas13 nucleic acid-guided nuclease. Notethat trans-cleavage activity is not triggered unless and untilcis-cleavage activity (i.e., sequence specific activity) is initiated.Nucleic acid-guided nucleases include Type V and Type VI nucleicacid-guided nucleases, as well as nucleic acid-guided nucleases thatcomprise a RuvC nuclease domain or a RuvC-like nuclease domain but lackan HNH nuclease domain. Nucleic acid-guided nucleases with theseproperties are reviewed in Makarova and Koonin, Methods Mol. Biol.,1311:47-75 (2015) and Koonin, et al., Current Opinion in Microbiology,37:67-78 (2020) and updated databases of nucleic acid-guided nucleasesand nuclease systems that include newly-discovered systems includeBioGRID ORCS (orcs:thebiogrid.org); GenomeCRISPR (genomecrispr.org);Plant Genome Editing Database (plantcrispr.org) and CRISPRCasFinder(crispercas.i2bc.paris-saclay.fr).

The type of nucleic acid-guided nuclease utilized in the method ofdetection depends on the type of target nucleic acid of interest to bedetected. For example, a DNA nucleic acid-guided nuclease (e.g., aCas12a, Cas14a, or Cas3) should be utilized if the target nucleic acidof interest is a DNA molecule, and an RNA nucleic acid-guided nuclease(e.g., Cas13a or Cas12g) should be utilized if the target nucleic acidof interest is an RNA molecule. Exemplary nucleic acid-guided nucleasesinclude, but are not limited to, Cas RNA-guided DNA nucleic acid-guidednucleases, such as Cas3, Cas12a (e.g., AsCas12a, LbCas12a), Cas12b,Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, and Cas12j; CasRNA-guided RNA nucleic acid-guided nucleases, such as Cas13a (LbaCas13,LbuCas13, LwaCas13), Cas13b (e.g., CccaCas13b, PsmCas13b), and Cas12g;and any other nucleic acid (DNA, RNA, or cDNA) targeting nucleicacid-guided nuclease with cis-cleavage activity and collateraltrans-cleavage activity. In some embodiments, the nucleic acid-guidednuclease is a Type V CRISPR-Cas nuclease, such as Cas12a, Cas13a, orCas14a. In some embodiments, the nucleic acid-guided nuclease is a TypeI CRISPR-Cas nuclease, such as Cas3. Type II and Type VI nucleicacid-guided nucleases may also be employed.

In an RNP with a single crRNA (i.e., lacking/without a tracrRNA), Cas12anucleases and related homologs and orthologs interact with a PAM(protospacer adjacent motif) sequence in a target nucleic acid for dsDNAunwinding and R-loop formation. Cas12a nucleases employ a multistepmechanism to ensure accurate recognition of spacer sequences in thetarget nucleic acid. The WED, REC1 and PAM-interacting (PI) domains ofCas12a nucleases are responsible for PAM recognition and for initiatinginvasion of the crRNA in the target dsDNA and for R-loop formation. Ithas been hypothesized that a conserved lysine residue is inserted intothe dsDNA duplex, possibly initiating template strand/non-templatestrand unwinding. (See Jinek, et al, Mol. Cell, 73(3):589-600.e4(2019).) PAM binding further introduces a kink in the target strand,which further contributes to local strand separation and facilitatesbase paring of the target strand to the seed segment of the crRNA whilethe displaced non-target strand is stabilized by interactions with thePAM-interacting domains. (Id.) The variant nucleic acid-guided nucleasesdisclosed herein and discussed in detail below have been engineered todisrupt one or both of the WED and PI domains to reconfigure the site ofunwinding and R-loop formation to, e.g., sterically obstruct dsDNAtarget nucleic acids from binding to the variant nucleic acid-guidednuclease and/or to minimize strand separation and/or stabilization ofthe non-target strand. Though contrary to common wisdom, engineering thevariant nucleic acid-guided nucleases in this way contributes to arobust and high-fidelity cascade assay.

The variant nucleic acid-guided nucleases disclosed herein are variantsof wildtype Type V nucleases LbCas12a (Lachnospriaceae bacteriumCas12a), AsCas 12a (Acidaminococcus sp. BV3L6 Cas12a), CtCas12a(Candidatus Methanoplasma termitum Cas12a), EeCas12a (Eubacteriumeligens Cas12a), Mb3Cas12a (Moraxella bovoculi Cas12a), FnCas12a(Francisella novicida Cas12a), FnoCas12a (Francisella tularensis subsp.novicida FTG Cas12a), FbCas 12a (Flavobacteriales bacterium Cas12a),Lb4Cas 12a (Lachnospira eligens Cas12a), MbCas12a (Moraxella bovoculiCas12a), Pb2Cas12a (Prevotella bryantii Cas12a), PgCas12a (CandidatusParcubacteria bacterium Cas12a), AaCas12a (Acidaminococcus sp. Cas12a),BoCas 12a (Bacteroidetes bacterium Cas12a), CMaCas 12a (CandidatusMethanomethylophilus alvus CMx1201 Cas12a), and to-be-discoveredequivalent Cas12a nucleic acid-guided nucleases and homologs andorthologs of these nucleic acid-guided nucleases (and other nucleicacid-guided nucleases that exhibit both cis-cleavage and trans-cleavageactivity), where mutations have been made to the PAM interacting domainssuch that double-stranded DNA (dsDNA) substrates are bound much moreslowly to the variant nucleic acid-guided nucleases than to theirwildtype nucleic acid-guided nuclease counterpart, yet single-strandedDNA (ssDNA) substrates are bound at the same rate or nearly so as theirwildtype nucleic acid-guided nuclease counterpart. The variant nucleicacid-guided nucleases comprise reconfigured domains that interact withthe PAM region or surrounding sequences on the blocked nucleic acidmolecules to achieve this phenotype and are described in detail below.

Guide RNA (gRNA)

The present disclosure detects a target nucleic acid of interest via areaction mixture containing at least two guide RNAs (gRNAs) eachincorporated into a different RNP complex (i.e., RNP1 and RNP2).Suitable gRNAs include at least one crRNA region to enable specificityin every reaction. The gRNA of RNP1 is specific to a target nucleic acidof interest and the gRNA of RNP2 is specific to an unblocked nucleicacid or a synthesized activating molecule (both described in detailbelow). As will be clear given the description below, an advantageousfeature of the cascade assay is that, with the exception of the gRNA inthe RNP1 (i.e., the gRNA specific to the target nucleic acid ofinterest), the cascade assay components can stay the same (i.e., areidentical or substantially identical) no matter what target nucleicacid(s) of interest are being detected, and the gRNA in RNP1 is easilyreprogrammable.

Like the nucleic acid-guided nuclease, the gRNA may be provided in thecascade assay reaction mix in a preassembled RNP, as an RNA molecule, ormay also be provided as a DNA sequence to be transcribed, in, e.g., avector backbone. Providing the gRNA in a pre-assembled RNP complex(i.e., RNP1 or RNP2) is preferred if rapid kinetics are preferred. Ifprovided as a gRNA molecule, the gRNA sequence may include multipleendoribonuclease recognition sites (e.g., Csy4) for multiplexprocessing. Alternatively, if provided as a DNA sequence to betranscribed, an endoribonuclease recognition site may be encoded betweenneighboring gRNA sequences such that more than one gRNA can betranscribed in a single expression cassette. Direct repeats can alsoserve as endoribonuclease recognition sites for multiplex processing.Guide RNAs are generally about 20 nucleotides to about 300 nucleotidesin length and may contain a spacer sequence containing a plurality ofbases and complementary to a protospacer sequence in the targetsequence. The gRNA spacer sequence may be 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 98%, 99%, or more complementary to its intended targetnucleic acid of interest.

The gRNA of RNP1 is capable of complexing with the nucleic acid-guidednuclease of RNP1 to perform cis-cleavage of a target nucleic acid ofinterest (e.g., a DNA or RNA), which triggers non-sequence specifictrans-cleavage of other molecules in the reaction mix. Guide RNAsinclude any polynucleotide sequence having sufficient complementaritywith a target nucleic acid of interest (or target sequences generated byunblocking blocked nucleic acid molecules or target sequences generatedby synthesizing synthesized activating molecules as described below).Target nucleic acids of interest (describe in detail above) preferablyinclude a protospacer-adjacent motif (PAM), and, following gRNA binding,the nucleic acid-guided nuclease induces a double-stranded break eitherinside or outside the protospacer region of the target nucleic acid ofinterest.

In some embodiments, the gRNA (e.g., of RNP1) is an exo-resistantcircular molecule that can include several DNA bases between the 5′ endand the 3′ end of a natural guide RNA and is capable of binding a targetsequence. The length of the circularized guide for RNP1 can be such thatthe circular form of guide can be complexed with a nucleic acid-guidednuclease to form a modified RNP1 which can still retain its cis-cleavagei.e., (specific) and trans-cleavage (i.e., non-specific) nucleaseactivity.

In any of the foregoing embodiments, the gRNA may be a modified ornon-naturally occurring nucleic acid molecule. In some embodiments, thegRNAs of the disclosure may further contain a locked nucleic acid (LNA),a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). Byway of further example, a modified nucleic acid molecule may contain amodified or non-naturally occurring nucleoside, nucleotide, and/orinternucleoside linkage, such as a 2′-O-methyl (2′-O-Me) modifiednucleoside, a 2′-fluoro (2′-F) modified nucleoside, and aphosphorothioate (PS) bond, or any other nucleic acid moleculemodifications described herein.

Ribonucleoprotein (RNP) Complex

As described above, although the cascade assay “reaction mix” maycomprise separate nucleic acid-guided nucleases and gRNAs (or codingsequences therefor), the cascade assays preferably comprise preassembledribonucleoprotein complexes (RNPs) in the reaction mix, allowing forfaster detection kinetics. The present cascade assay employs at leasttwo types of RNP complexes—RNP1 and RNP2—each type containing a nucleicacid-guided nuclease and a gRNA. RNP1 and RNP2 may comprise the samenucleic acid-guided nuclease or may comprise different nucleicacid-guided nucleases; however, the gRNAs in RNP1 and RNP2 are differentand are configured to detect different nucleic acids. In someembodiments, the reaction mixture contains about 1 fM to about 10 μM ofa given RNP1, or about 1 μM to about 1 μM of a given RNP1, or about 10μM to about 500 μM of a given RNP1. In some embodiments the reactionmixture contains about 6×10⁴ to about 6×10¹² complexes per microliter(μl) of a given RNP1, or about 6×10⁶ to about 6×10¹⁰ complexes permicroliter (μl) of a given RNP1. In some embodiments, the reactionmixture contains about 1 fM to about 500 μM of a given RNP2, or about 1μM to about 250 μM of a given RNP2, or about 10 μM to about 100 μM of agiven RNP2. In some embodiments the reaction mixture contains about6×10⁴ to about 6×10¹² complexes per microliter (μl) of a given RNP2 orabout 6×10⁶ to about 6×10¹² complexes per microliter (μl) of a givenRNP2. See Example II below describing preassembling RNPs and Examples Vand VI below describing various cascade assay conditions where therelative concentrations of RNP2 and the blocked nucleic acid moleculesis adjusted as described below.

In any of the embodiments of the disclosure, the reaction mixtureincludes 1 to about 1,000 different RNP1s (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 19, 20, 21, 22, 23, 24, 25, 50,75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, or 1,0000 or more RNP1s), wheredifferent RNP1s comprise a different gRNA (or crRNA thereof)polynucleotide sequence. For example, a reaction mixture designed forenvironmental or oncology testing comprises more than one uniqueRNP1-gRNA (or RNP1-crRNA) ribonucleoprotein complex for the purpose ofdetecting more than one target nucleic acid of interest. That is, morethan one RNP1 may also be present for the purpose of targeting onetarget nucleic acid of interest from many sources or for targeting morethan one target nucleic acid of interest from a single source.

In any of the foregoing embodiments, the gRNA of RNP1 may be homologousor heterologous, relative to the gRNA of other RNP1(s) present in thereaction mixture. A homologous mixture of RNP1 gRNAs has a number ofgRNAs with the same nucleotide sequence, whereas a heterologous mixtureof RNP1 gRNAs has multiple gRNAs with different nucleotide sequences(e.g., gRNAs targeting different loci, genes, variants, and/or microbialspecies). Therefore, the disclosed methods of identifying one or moretarget nucleic acids of interest may include a reaction mixturecontaining more than two heterologous gRNAs, more than threeheterologous gRNAs, more than four heterologous gRNAs, more than fiveheterologous gRNAs, more than six heterologous gRNAs, more than sevenheterologous gRNAs, more than eight heterologous gRNAs, more than nineheterologous gRNAs, more than ten heterologous gRNAs, more than elevenheterologous gRNAs, more than twelve heterologous gRNAs, more thanthirteen heterologous gRNAs, more than fourteen heterologous gRNAs, morethan fifteen heterologous gRNAs, more than sixteen heterologous gRNAs,more than seventeen heterologous gRNAs, more than eighteen heterologousgRNAs, more than nineteen heterologous gRNAs, more than twentyheterologous gRNAs, more than twenty-one heterologous gRNAs, more thantwenty-three heterologous gRNAs, more than twenty-four heterologousgRNAs, or more than twenty-five heterologous gRNAs. Such a heterologousmixture of RNP1 gRNAs in a single reaction enables multiplex testing.

As a first non-limiting example of a heterologous mixture of RNP1 gRNAs,the reaction mixture may contain: a number of RNP1s (RNP1-1s) having agRNA targeting parainfluenza virus 1; a number of RNP1s (RNP1-2s) havinga gRNA targeting human metapneumovirus; a number of RNP1s (RNP1-3s)having a gRNA targeting human rhinovirus; a number of RNP1s (RNP1-4s)having a gRNA targeting human enterovirus; and a number of RNP1s(RNP1-5s) having a gRNA targeting coronavirus HKU1. As a secondnon-limiting example of a heterologous mixture of RNP1 gRNAs, thereaction mixture may contain: a number of RNP1s containing a gRNAtargeting two or more SARS—Co-V-2 variants, e.g., B.1.1.7, B.1.351, P.1,B.1.617.2, BA.1, BA.2, BA.2.12.1, BA.4, and BA.5 and subvariantsthereof.

As another non-limiting example of a heterologous mixture of RNP1 gRNAs,the reaction mixture may contain RNP1s targeting two or more targetnucleic acids of interest from organisms that infect grapevines, such asGuignardia bidwellii (RNP1-1), Uncinula necator (RNP1-2), Botrytiscincerea (RNP1-3), Plasmopara viticola (RNP1-4), and Botryotinisfuckleina (RNP1-5).

Reporter Moieties

The cascade assay detects a target nucleic acid of interest viadetection of a signal generated in the reaction mix by a reportermoiety. In some embodiments the detection of the target nucleic acid ofinterest occurs virtually instantaneously. For example, see the resultsreported in Example VI for assays comprising 3e4 or 30 copies of MRSAtarget and within 1 minute or less at 3 copies of MRSA target (see,e.g., FIGS. 10B-10H). Reporter moieties can comprise DNA, RNA, a chimeraof DNA and RNA, and can be single stranded, double stranded, or a moietythat is a combination of single stranded portions and double strandedportions.

Depending on the type of reporter moiety used, trans- and/orcis-cleavage by the nucleic acid-guided nuclease in RNP2 releases asignal. In some embodiments, trans-cleavage of stand-alone reportermoieties (e.g., not bound to any blocked nucleic acid molecules orblocked primer molecules) may generate signal changes at rates that areproportional to the cleavage rate, as new RNP2s are activated over time(shown in FIG. 1B and at top of FIG. 4 ). Trans-cleavage by either anactivated RNP1 or an activated RNP2 may release a signal. In alternativeembodiments and preferably, the reporter moiety may be bound to theblocked nucleic acid molecule, where trans-cleavage of the blockednucleic acid molecule (or blocked primer molecule) and conversion to anunblocked nucleic acid molecule (or unblocked primer molecule) maygenerate signal changes at rates that are proportional to the cleavagerate, as new RNP2s are activated over time, thus allowing for real timereporting of results (shown at FIG. 4 , center). In yet anotherembodiment, the reporter moiety may be bound to a blocked nucleic acidmolecule such that cis-cleavage following the binding of the RNP2 to anunblocked nucleic acid molecule releases a PAM distal sequence, which inturn generates a signal at rates that are proportional to the cleavagerate (shown at FIG. 4 , bottom). In this case, activation of RNP2 bycis-(target specific) cleavage of the unblocked nucleic acid moleculedirectly produces a signal, rather than producing a signal viaindiscriminate trans-cleavage activity. Alternatively or in addition, areporter moiety may be bound to the gRNA.

The reporter moiety may be a synthetic molecule linked or conjugated toa reporter and quencher such as, for example, a TaqMan probe with a dyelabel (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end.The reporter and quencher may be about 20-30 bases apart or less (i.e.,10-11 nm apart or less) for effective quenching via fluorescenceresonance energy transfer (FRET). Alternatively, signal generation mayoccur through different mechanisms. Other detectable moieties, labels,or reporters can also be used to detect a target nucleic acid ofinterest as described herein. Reporter moieties can be labeled in avariety of ways, including direct or indirect attachment of a detectablemoiety such as a fluorescent moiety, hapten, or colorimetric moiety.

Examples of detectable moieties include various radioactive moieties,enzymes, prosthetic groups, fluorescent markers, luminescent markers,bioluminescent markers, metal particles, and protein-protein bindingpairs, e.g., protein-antibody binding pairs. Examples of fluorescentmoieties include, but are not limited to, yellow fluorescent protein(YFP), green fluorescence protein (GFP), cyan fluorescence protein(CFP), umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, cyanines, dansylchloride, phycocyanin, and phycoerythrin. Examples of bioluminescentmarkers include, but are not limited to, luciferase (e.g., bacterial,firefly, click beetle and the like), luciferin, and aequorin. Examplesof enzyme systems having visually detectable signals include, but arenot limited to, galactosidases, glucorinidases, phosphatases,peroxidases, and cholinesterases. Identifiable markers also includeradioactive elements such as ¹²⁵I, ³⁵S, ¹⁴C, or ³H. Reporters can alsoinclude a change in pH or charge of the cascade assay reaction mix.

The methods used to detect the generated signal will depend on thereporter moiety or moieties used. For example, a radioactive label canbe detected using a scintillation counter, photographic film as inautoradiography, or storage phosphor imaging. Fluorescent labels can bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence can bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Enzymatic labels can be detected byproviding the appropriate substrates for the enzyme and detecting theresulting reaction product. Simple colorimetric labels can be detectedby observing the color associated with the label. When pairs offluorophores are used in an assay, fluorophores are chosen that havedistinct emission patterns (wavelengths) so that they can be easilydistinguished. In some embodiments, the signal can be detected bylateral flow assays (LFAs). Lateral flow tests are simple devicesintended to detect the presence or absence of a target nucleic acid ofinterest in a sample. LFAs can use nucleic acid molecules conjugatednanoparticles (often gold, e.g., RNA-AuNPs or DNA-AuNPs) as a detectionprobe, which hybridizes to a complementary target sequence. (See FIG. 9and the description thereof below.) The classic example of an LFA is thehome pregnancy test.

Single-stranded, double-stranded or reporter moieties comprising bothsingle- and double-stranded portions can be introduced to show a signalchange proportional to the cleavage rate, which increases with every newactivated RNP2 complex over time. In some embodiments and as describedin detail below, reporter moieties can also be embedded into the blockednucleic acid molecules (or blocked primer molecules) for real timereporting of results.

For example, the method of detecting a target nucleic acid molecule in asample using a cascade assay as described herein can involve contactingthe reaction mix with a labeled detection ssDNA containing a fluorescentresonance energy transfer (FRET) pair, a quencher/phosphor pair, orboth. A FRET pair consists of a donor chromophore and an acceptorchromophore, where the acceptor chromophore may be a quencher molecule.FRET pairs (donor/acceptor) suitable for use include, but are notlimited to, EDANS/fluorescein, IAEDANS/fluorescein,fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL,fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5, TexasRed/DABCYL, BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL, andfluorescein/LC Red 705. In addition, a fluorophore/quantum dotdonor/acceptor pair can be used. EDANS is(5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid); IAEDANS is5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid);DABCYL is 4-(4-dimethylaminophenyl) diazenylbenzoic acid. Usefulquenchers include, but are not limited to, BHQ, DABCYL, QSY 7 and QSY33.

In any of the foregoing embodiments, the reporter moiety may compriseone or more modified nucleic acid molecules, containing a modifiednucleoside or nucleotide. In some embodiments the modified nucleoside ornucleotide is chosen from 2′-(2′-O-Me) modified nucleoside, a 2′-fluoro(2′-F) modified nucleoside, and a phosphorothioate (PS) bond, or anyother nucleic acid molecule modifications described below.

Nucleic Acid Modifications

For any of the nucleic acid molecules described herein (e.g., blockednucleic acid molecules, blocked primer molecules, gRNAs, templatemolecules, synthesized activating molecules, and reporter moieties), thenucleic acid molecules may be used in a wholly or partially modifiedform. Typically, modifications to the blocked nucleic acid molecules,gRNAs, template molecules, reporter moieties, and blocked primermolecules described herein are introduced to optimize the molecule'sbiophysical properties (e.g., increasing nucleic acid-guided nucleaseresistance and/or increasing thermal stability). Modifications typicallyare achieved by the incorporation of, for example, one or morealternative nucleosides, alternative sugar moieties, and/or alternativeinternucleoside linkages.

For example, one or more of the cascade assay components may include oneor more of the following nucleoside modifications: 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine and other alkynylderivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, and/or 3-deazaguanine and 3-deazaadenine. The nucleicacid molecules described herein (e.g., blocked nucleic acid molecules,blocked primer molecules, gRNAs, reporter molecules, synthesizedactivating molecules, and template molecules) may also includenucleobases in which the purine or pyrimidine base is replaced withother heterocycles, for example 7-deaza-adenine, 7-deazaguanosine,2-aminopyridine, and/or 2-pyridone. Further modification of the nucleicacid molecules described herein may include nucleobases disclosed inU.S. Pat. No. 3,687,808; Kroschwitz, ed., The Concise Encyclopedia ofPolymer Science and Engineering, NY, John Wiley & Sons, 1990, pp.858-859; Englisch, et al., Angewandte Chemie, 30:613 (1991); andSanghvi, Chapter 16, Antisense Research and Applications, CRC Press,Gait, ed., 1993, pp. 289-302.

In addition to or as an alternative to nucleoside modifications, thecascade assay components may comprise 2′ sugar modifications, including2′-O-methyl (2′-O-Me), 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE), 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, and/or2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylamino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂OCH₂N(CH₃)₂. Other possible 2′-modifications that can modify thenucleic acid molecules described herein (i.e., blocked nucleic acidmolecules, gRNAs, synthesized activating molecules, reporter molecules,and blocked primer molecules) may include all possible orientations ofOH; F; S-, or N-alkyl (mono- or di-); O-, S-, or N-alkenyl (mono- ordi-); O-, S- or N-alkynyl (mono- or di-); or O-alkyl-O-alkyl, whereinthe alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 toC10 alkyl or C2 to C10 alkenyl and alkynyl. Other potential sugarsubstituent groups include, e.g., aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl(—CH₂—CH═CH₂), —O-allyl (—O—CH₂—CH═CH₂) and fluoro (F). 2′-sugarsubstituent groups may be in the arabino (up) position or ribo (down)position. In some embodiments, the 2′-arabino modification is 2′-F.Similar modifications may also be made at other positions on theinterfering RNA molecule, particularly the 3′ position of the sugar onthe 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the5′ position of 5′ terminal nucleotide. Oligonucleotides may also havesugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar.

Finally, modifications to the cascade assay components may compriseinternucleoside modifications such as phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates, and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.

The Signal Boosting Cascade Assay Employing Blocked Nucleic AcidMolecules

Before getting to the details relating to addressing undesired unwindingof the blocked nucleic acid molecules (or blocked primer molecules),understanding the cascade assay itself is key. FIG. 1B, described above,depicts the cascade assay generally. A specific embodiment of thecascade assay utilizing blocked nucleic acid molecules is depicted inFIG. 2A and described in detail below. In this embodiment, a blockednucleic acid is used to prevent the activation of RNP2 in the absence ofa target nucleic acid of interest. The method in FIG. 2A begins withproviding the cascade assay components RNP1 (201), RNP2 (202) andblocked nucleic acid molecules (203). RNP1 (201) comprises a gRNAspecific for a target nucleic acid of interest and a nucleic acid-guidednuclease (e.g., Cas 12a or Cas 14 for a DNA target nucleic acid ofinterest or a Cas 13a for an RNA target nucleic acid of interest) andRNP2 (202) comprises a gRNA specific for an unblocked nucleic acidmolecule and a nucleic acid-guided nuclease (again, e.g., Cas 12a or Cas14 for a DNA unblocked nucleic acid molecule or a Cas 13a for an RNAunblocked nucleic acid molecule). As described above, the nucleicacid-guided nucleases in RNP1 (201) and RNP2 (202) can be the same ordifferent depending on the type of target nucleic acid of interest andunblocked nucleic acid molecule. What is key, however, is that thenucleic acid-guided nucleases in RNP1 and RNP2 may be activated to havetrans-cleavage activity following initiation of cis-cleavage activity.

In a first step, a sample comprising a target nucleic acid of interest(204) is added to the cascade assay reaction mix. The target nucleicacid of interest (204) combines with and activates RNP1 (205) but doesnot interact with or activate RNP2 (202). Once activated, RNP1 binds thetarget nucleic acid of interest (204) and cuts the target nucleic acidof interest (204) via sequence-specific cis-cleavage, activatingnon-specific trans-cleavage of other nucleic acids present in thereaction mix, including the blocked nucleic acid molecules (203). Atleast one of the blocked nucleic acid molecules (203) becomes anunblocked nucleic acid molecule (206) when the blocking moiety (207) isremoved. As described below, “blocking moiety” may refer to nucleosidemodifications, topographical configurations such as secondarystructures, and/or structural modifications.

Once at least one of the blocked nucleic acid molecules (203) isunblocked, the unblocked nucleic acid molecule (206) can then bind toand activate an RNP2 (208). Because the nucleic acid-guided nucleases inthe RNP1s (205) and RNP2s (208) have both cis- and trans-cleavageactivity, the trans-cleavage activity causes more blocked nucleic acidmolecules (203) become unblocked nucleic acid molecules (206) triggeringactivation of even more RNP2s (208) and more trans-cleavage activity ina cascade. FIG. 2A at bottom depicts the concurrent activation ofreporter moieties. Intact reporter moieties (209) comprise a quencher(210) and a fluorophore (211) linked by a nucleic acid sequence. Asdescribed above in relation to FIG. 1B, the reporter moieties are alsosubject to trans-cleavage by activated RNP1 (205) and RNP2 (208). Theintact reporter moieties (209) become activated reporter moieties (212)when the quencher (210) is separated from the fluorophore (211),emitting a fluorescent signal (213). Signal strength increases rapidlyas more blocked nucleic acid molecules (203) become unblocked nucleicacid molecules (206) triggering cis-cleavage activity of more RNP2s(208) and thus more trans-cleavage activity of the reporter moieties(209). Again, the reporter moieties are shown here as separate moleculesfrom the blocked nucleic acid molecules, but other configurations may beemployed and are discussed in relation to FIG. 4 . One particularlyadvantageous feature of the cascade assay is that, with the exception ofthe gRNA in the RNP1 (gRNA1), the cascade assay components are modularin the sense that the components stay the same no matter what targetnucleic acid(s) of interest are being detected.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid molecule(220) and an exemplary technique for unblocking the blocked nucleic acidmolecules described herein. A blocked single-stranded ordouble-stranded, circular or linear, DNA or RNA molecule (220)comprising a target strand (222) may contain a partial hybridizationwith a complementary non-target strand nucleic acid molecule (224)containing unhybridized and cleavable secondary loop structures (226)(e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissinghairpins, internal loops, bulges, and multibranch loops). Trans-cleavageof the loops by, e.g., activated RNP1s or RNP2s, generates short strandnucleotide sequences or regions (228) which, because of the short lengthand low melting temperature T m can dehybridize at room temperature(e.g., 15°-25° C.), thereby unblocking the blocked nucleic acid molecule(220) to create an unblocked nucleic acid molecule (230), enabling theinternalization of the unblocked nucleic acid molecule (230) (targetstrand) into an RNP2, leading to RNP2 activation.

A blocked nucleic acid molecule may be single-stranded ordouble-stranded, circular or linear, and may further contain a partiallyhybridized nucleic acid sequence containing cleavable secondary loopstructures, as exemplified by “L” in FIGS. 2C-2E. Such blocked nucleicacid molecules typically have a low binding affinity, or highdissociation constant (K_(d)) in relation to binding to RNP2 and may bereferred to herein as a high K_(d) nucleic acid molecule. In the contextof the present disclosure, the binding of blocked or unblocked nucleicacid molecules or blocked or unblocked primer molecules to RNP2, lowK_(d) values range from about 100 fM to about 1 aM or lower (e.g., 100zM) and high K_(d) values are in the range of 100 nM to about 10-100 10mM and thus are about 10⁵-, 10⁶-, 10⁷-, 10⁸-, 10⁹- to 10¹⁰-fold orhigher as compared to low K_(d) values. Of course, the ideal blockednucleic acid molecule would have an “infinite K_(d).”

The blocked nucleic acid molecules (high K_(d) molecules) describedherein can be converted into unblocked nucleic acid molecules (low K_(d)molecules—also in relation to binding to RNP2) via cleavage ofnuclease-cleavable regions (e.g., via active RNP1s and RNP2s). Theunblocked nucleic acid molecule has a higher binding affinity for thegRNA in RNP2 than does the blocked nucleic acid molecule, although, asdescribed below, there is some “leakiness” where some blocked nucleicacid molecules are able to interact with the gRNA in the RNP2 triggeringundesired unwinding.

Once the unblocked nucleic acid molecule is bound to RNP2, the RNP2activation triggers trans-cleavage activity, which in turn leads to moreRNP2 activation by further cleaving blocked nucleic acid molecules,resulting in a positive feedback loop or cascade.

In embodiments where blocked nucleic acid molecules are linear and/orform a secondary structure, the blocked nucleic acid molecules may besingle-stranded (ss) or double-stranded (ds) and contain a firstnucleotide sequence and a second nucleotide sequence. The firstnucleotide sequence has sufficient complementarity to hybridize to agRNA of RNP2, and the second nucleotide sequence does not. The first andsecond nucleotide sequences of a blocked nucleic acid molecule may be onthe same nucleic acid molecule (e.g., for single-strand embodiments) oron separate nucleic acid molecules (e.g., for double-strandembodiments). Trans-cleavage (e.g., via RNP1 or RNP2) of the secondnucleotide sequence converts the blocked nucleic acid molecule to asingle-strand unblocked nucleic acid molecule. The unblocked nucleicacid molecule contains only the first nucleotide sequence, which hassufficient complementarity to hybridize to the gRNA of RNP2, therebyactivating the trans-cleavage activity of RNP2.

In some embodiments, the second nucleotide sequence at least partiallyhybridizes to the first nucleotide sequence, resulting in a secondarystructure containing at least one loop (e.g., hairpin loops, tetraloops,pseudoknots, junctions, kissing hairpins, internal loops, bulges, andmultibranch loops). Such loops block the nucleic acid molecule frombinding or incorporating into an RNP complex thereby initiating cis- ortrans-cleavage (see, e.g., the exemplary structures in FIGS. 2C-2F).

In some embodiments, the blocked nucleic acid molecule may contain aprotospacer adjacent motif (PAM) sequence, or partial PAM sequence,positioned between the first and second nucleotide sequences, where thefirst sequence is 5′ to the PAM sequence, or partial PAM sequence, (seeFIG. 2G). Inclusion of a PAM sequence may increase the reaction kineticsinternalizing the unblocked nucleic acid molecule into RNP2 and thusdecrease the time to detection. In other embodiments, the blockednucleic acid molecule does not contain a PAM sequence.

In some embodiments, the blocked nucleic acid molecules (i.e., highK_(d) nucleic acid molecules in relation to binding to RNP2) of thedisclosure may include a structure represented by Formula I (e.g., FIG.2C), Formula II (e.g., FIG. 2D), Formula III (e.g., FIG. 2E), or FormulaIV (e.g., FIG. 2F) wherein Formulas I-IV are in the 5′-to-3′ direction:

A-(B-L)J-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;    -   B is 4-12 nucleotides in length;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10;    -   C is 4-15 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then A-(B-L)J-C and T-D are separate nucleic acid        strands;    -   T is 17-135 nucleotides in length (e.g., 17-100, 17-50, or        17-25) and comprises a sequence complementary to B and C; and    -   D is 0-10 nucleotides in length and comprises a sequence        complementary to A;

D-T-T′-C-(L-B)J-A  (Formula II);

wherein D is 0-10 nucleotides in length;

-   -   T-T′ is 17-135 nucleotides in length (e.g., 17-100, 17-50, or        17-25);    -   T′ is 1-10 nucleotides in length and does not hybridize with T;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T;    -   L is 3-25 nucleotides in length and does not hybridize with T;    -   B is 4-12 nucleotides in length and comprises a sequence        complementary to T;    -   J is an integer between 1 and 10;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D;

T-D-M-A-(B-L)J-C  (Formula III);

-   -   wherein T is 17-135 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-10 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then T-D and    -   A-(B-L)J-C are separate nucleic acid strands;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D;    -   B is 4-12 nucleotides in length and comprises a sequence        complementary to T;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10; and    -   C is 4-15 nucleotides in length;

T-D-M-A-Lp-C  (Formula IV);

wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50, or17-25);

-   -   D is 0-15 nucleotides in length;    -   M is 1-25 nucleotides in length;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D; and    -   L is 3-25 nucleotides in length;    -   p is 0 or 1;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T.        In alternative embodiments of any of these molecules, T (or        T-T′) can have a maximum length of 1000 nucleotides, e.g., at        most 750, at most 500, at most 400, at more 300, at most 250, at        most 200, at most 150, at most 135, at most 100, at most 75, at        most 50, or at most 25 nucleotides.

Nucleotide mismatches can be introduced in any of the above structurescontaining double-strand segments (for example, where M is absent inFormula I or Formula III) to reduce the melting temperature (T m) of thesegment such that once the loop (L) is cleaved, the double-strandsegment is unstable and dehybridizes rapidly. The percentage ofnucleotide mismatches of a given segment may vary between 0% and 50%;however, the maximum number of nucleotide mismatches is limited to anumber where the secondary loop structure still forms. “Segments” in theabove statement refers to A, B, and C. In other words, the number ofhybridized bases can be less than or equal to the length of eachdouble-strand segment and vary based on number of mismatches introduced.

In any blocked nucleic acid molecule having the structure of Formula I,III, or IV, T will have sequence complementarity to a nucleotidesequence (e.g., a spacer sequence) within a gRNA of RNP2. The nucleotidesequence of T is to be designed such that hybridization of T to the gRNAof RNP2 activates the trans-nuclease activity of RNP2. In any blockednucleic acid molecule having structure of Formula II, T-T′ will havesequence complementarity to a sequence (e.g., a spacer sequence) withinthe gRNA of RNP2. The nucleotide sequence of T-T′ is to be designed suchthat hybridization of T-T′ to the gRNA of RNP2 activates thetrans-nuclease activity of RNP2. For T or T-T′, full complementarity tothe gRNA is not necessarily required, provided there is sufficientcomplementarity to cause hybridization and trans-cleavage activation ofRNP2.

In any of the foregoing embodiments, the blocked nucleic acid moleculesof the disclosure may and preferably do further contain a reportermoiety attached thereto such that cleavage of the blocked nucleic acidreleases a signal from the reporter moiety. (See FIG. 4 , mechanismsdepicted at center and bottom.)

Also, in any of the foregoing embodiments, the blocked nucleic acidmolecule may be a modified or non-naturally occurring nucleic acidmolecule. In some embodiments, the blocked nucleic acid molecules of thedisclosure may further contain a locked nucleic acid (LNA), a bridgednucleic acid (BNA), and/or a peptide nucleic acid (PNA). The blockednucleic acid molecule may contain a modified or non-naturally occurringnucleoside, nucleotide, and/or internucleoside linkage, such as a2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modifiednucleoside, and a phosphorothioate (PS) bond, any other nucleic acidmolecule modifications described above, and any combination thereof.

FIG. 2G at left shows an exemplary single-strand blocked nucleic acidmolecule and how the configuration of this blocked nucleic acid moleculeis able to prevent (or significantly prevent) undesired unwinding of theblocked nucleic acid molecule (or blocked primer molecule) and R-loopformation with an RNP complex, thereby blocking activation of thetrans-cleavage activity of RNP2. The single-strand blocked nucleic acidmolecule is self-hybridized and comprises: a target strand (TS) sequencecomplementary to the gRNA (e.g., crRNA) of RNP2; a cleavable non-targetstrand (NTS) sequence that is partially hybridized (e.g., it containssecondary loop structures) to the TS sequence; and a protospaceradjacent motif (PAM) sequence (e.g., 5′ NAAA 3′) that is specificallylocated at the 3′ end of the TS sequence. An RNP complex with 3′→5′diffusion (e.g., 1D diffusion) initiates R-loop formation upon PAMrecognition. R-loop formation is completed upon a stabilizing ≥17 basehybridization of the TS to the gRNA of RNP2; however, because of theorientation of the PAM sequence relative to the secondary loopstructure(s), the blocked nucleic acid molecule sterically prevents thetarget strand from hybridizing with the gRNA of RNP2, thereby blockingthe stable R-loop formation required for the cascade reaction.

FIG. 2G at right shows the blocked nucleic acid molecule being unblockedvia trans-cleavage (e.g., by RNP1) and subsequent dehybridization of thenon-target strand's secondary loop structures, followed by binding ofthe target strand to the gRNA of RNP2, thereby completing stable R-loopformation and activating the trans-cleavage activity of the RNP2complex.

In some embodiments, the blocked nucleic acid molecules provided hereinare circular DNAs, RNAs or chimeric (DNA-RNA) molecules (FIG. 2H), andthe blocked nucleic acid molecules may include different basecompositions depending on the Cas enzyme used for RNP1 and RNP2. For thecircular design of blocked nucleic acid molecules, the 5′ and 3′ endsare covalently linked together. This configuration makes internalizationof the blocked nucleic acid molecule into RNP2—and subsequent RNP2activation—sterically unfavorable, thereby blocking the progression ofthe cascade assay. Thus, RNP2 activation (e.g., trans-cleavage activity)happens after cleavage of a portion of the blocked nucleic acid moleculefollowed by linearization and internalization of unblocked nucleic acidmolecule into RNP2.

In some embodiments, the blocked nucleic acid molecules aretopologically circular molecules with 5′ and 3′ portions hybridized toeach other using DNA, RNA, LNA, BNA, or PNA bases which have a very highmelting temperature (Tm). The high Tm causes the structure toeffectively behave as a circular molecule even though the 5′ and 3′ endsare not covalently linked. The 5′ and 3′ ends can also have basenon-naturally occurring modifications such as phosphorothioate bonds toprovide increased stability.

In embodiments where the blocked nucleic acid molecules are circularized(e.g., circular or topologically circular), as illustrated in FIG. 2H,each blocked nucleic acid molecule includes a first region, which is atarget sequence specific to the gRNA of RNP2, and a second region, whichis a sequence that can be cleaved by nuclease enzymes of activated RNP1and/or RNP2. The first region may include a nuclease-resistant nucleicacid sequence such as, for example, a phosphorothioate group or othernon-naturally occurring nuclease-resistant base modifications, forprotection from trans-nucleic acid-guided nuclease activity. In someembodiments, when the Cas enzyme in both RNP1 and RNP2 is Cas12a, thefirst region of the blocked nucleic acid molecule includes anuclease-resistant DNA sequence, and the second region of the blockednucleic acid molecule includes a cleavable DNA sequence. In otherembodiments, when the Cas enzyme in RNP1 is Cas12a and the Cas enzyme inRNP2 is Cas13a, the first region of the blocked nucleic acid moleculeincludes a nuclease-resistant RNA sequence, and the second region of theblocked nucleic acid molecule includes a cleavable DNA sequence and acleavable RNA sequence. In yet other embodiments, when the Cas enzyme inRNP1 is Cas13a and the Cas enzyme in RNP2 is Cas12a, the first region ofthe blocked nucleic acid molecule includes a nuclease-resistant DNAsequence, and the second region of the blocked nucleic acid moleculeincludes a cleavable DNA sequence and a cleavable RNA sequence. In someother embodiments, when the Cas enzyme in both RNP1 and RNP2 is Cas13a,the first region of the blocked nucleic acid molecule includes anuclease-resistant RNA sequence, and the second region of the blockednucleic acid molecule includes a cleavable RNA sequence.

The Signal Boosting Cascade Assay Employing Blocked Primer Molecules

The blocked nucleic acid molecules described above may also be blockedprimer molecules. Blocked primer molecules include a sequencecomplementary to a primer binding domain (PBD) on a template molecule(see description below in reference to FIGS. 3A and 3B) and can have thesame general structures as the blocked nucleic acid molecules describedabove. A PBD serves as a nucleotide sequence for primer hybridizationfollowed by primer polymerization by a polymerase. In any of Formulas I,II, or III described above, the blocked primer nucleic acid molecule mayinclude a sequence complementary to the PBD on the 5′ end of T. Theunblocked primer nucleic acid molecule can bind to a template moleculeat the PBD and copy the template molecule via polymerization by apolymerase.

Specific embodiments of the cascade assay which utilize blocked primermolecules and are depicted in FIGS. 3A and 3B. In the embodiments usingblocked nucleic acid molecules described above, activation of RNP1 bybinding of N nucleotides of the target nucleic acid molecules orcis-cleavage of the target nucleic acid molecules initiatestrans-cleavage of the blocked nucleic acid molecules which were used toactivate RNP2—that is, the unblocked nucleic acid molecules are a targetsequence for the gRNA in RNP2. In contrast, in the embodiments usingblocked primers activation of RNP1 and trans-cleavage unblocks a blockedprimer molecule that is then used to prime a template molecule forextension by a polymerase, thereby synthesizing synthesized activatingmolecules that are the target sequence for the gRNA in RNP2.

FIG. 3A is a diagram showing the sequence of steps in an exemplarycascade assay involving circular blocked primer molecules and lineartemplate molecules. At left of FIG. 3A is a cascade assay reaction mixcomprising 1) RNP1s (301) (only one RNP1 is shown); 2) RNP2s (302); 3)linear template molecules (330) (which is the non-target strand); 4) acircular blocked primer molecule (334) (i.e., a high K_(d) molecule);and 5) a polymerase (338), such as a 129 polymerase. The linear templatemolecule (330) (non-target strand) comprises a PAM sequence (331), aprimer binding domain (PBD) (332) and, optionally, a nucleosidemodification (333) to protect the linear template molecule (330) from3′→5′ exonuclease activity. Blocked primer molecule (334) comprises acleavable region (335) and a complement to the PBD (332) on the lineartemplate molecule (330).

Upon addition of a sample comprising a target nucleic acid of interest(304) (capable of complexing with the gRNA in RNP1 (301)), the targetnucleic acid of interest (304) is bound by with and activates RNP1 (305)but does not interact with or activate RNP2 (302). Once activated, RNP1cuts the target nucleic acid of interest (304) via sequence specificcis-cleavage, which activates non-specific trans-cleavage of othernucleic acids present in the reaction mix, including at least one of theblocked primer molecules (334). The circular blocked primer molecule(334) (i.e., a high K_(d) molecule, where high K_(d) relates to bindingto RNP2) upon cleavage becomes an unblocked linear primer molecule (344)(a low K_(d) molecule, where low K_(d) relates to binding to RNP2),which has a region (336) complementary to the PBD (332) on the lineartemplate molecule (330) and can bind to the linear template molecule(330).

Once the unblocked linear primer molecule (344) and the linear templatemolecule (330) are hybridized (i.e., hybridized at the PBD (332) of thelinear template molecule (330) and the PBD complement (336) on theunblocked linear primer molecule (344)), 3′→5′ exonuclease activity ofthe polymerase (338) removes the unhybridized single-stranded DNA at theend of the unblocked primer molecule (344) and the polymerase (338) cancopy the linear template molecule (330) to produce a synthesizedactivating molecule (346) which is a complement of the non-targetstrand, which is the target strand. The synthesized activating molecule(346) is capable of activating RNP2 (302→308). As described above,because the nucleic acid-guided nuclease in the RNP2 (308) complexexhibits (that is, possesses) both cis- and trans-cleavage activity,more blocked primer molecules (334) become unblocked primer molecules(344) triggering activation of more RNP2s (308) and more trans-cleavageactivity in a cascade. As stated above in relation to blocked andunblocked nucleic acid molecules (both linear and circular), theunblocked primer molecule has a higher binding affinity for the gRNA inRNP2 than does the blocked primer molecule, although there may be some“leakiness” where some blocked primer molecules are able to interactwith the gRNA in RNP2. However, an unblocked primer molecule has asubstantially higher likelihood than a blocked primer molecule tohybridize with the gRNA of RNP2.

FIG. 3A at bottom depicts the concurrent activation of reportermoieties. Intact reporter moieties (309) comprise a quencher (310) and afluorophore (311). As described above in relation to FIG. 1B, thereporter moieties are also subject to trans-cleavage by activated RNP1(305) and RNP2 (308). The intact reporter moieties (309) becomeactivated reporter moieties (312) when the quencher (310) is separatedfrom the fluorophore (311), and the fluorophore emits a fluorescentsignal (313). Signal strength increases rapidly as more blocked primermolecules (334) become unblocked primer molecules (344) generatingsynthesized activating molecules (346) and triggering activation of moreRNP2 (308) complexes and more trans-cleavage activity of the reportermoieties (309). Again, here the reporter moieties are shown as separatemolecules from the blocked nucleic acid molecules, but otherconfigurations may be employed and are discussed in relation to FIG. 4 .Also, as with the cascade assay embodiment utilizing blocked nucleicacid molecules that are not blocked primers, with the exception of thegRNA in RNP1, the cascade assay components stay the same no matter whattarget nucleic acid(s) of interest are being detected.

FIG. 3B is a diagram showing the sequence of steps in an exemplarycascade assay involving circular blocked primer molecules and circulartemplate molecules. The cascade assay of FIG. 3B differs from thatdepicted in FIG. 3A by the configuration of the template molecule. Wherethe template molecule in FIG. 3A was linear, in FIG. 3B the templatemolecule is circular. At left of FIG. 3B is a cascade assay reaction mixcomprising 1) RNP1s (301) (only one RNP1 is shown); 2) RNP2s (302); 3) acircular template molecule (352) (non-target strand); 4) a circularblocked primer molecule (334); and 5) a polymerase (338), such as a Φ29polymerase. The circular template molecule (352) (non-target strand)comprises a PAM sequence (331) and a primer binding domain (PBD) (332).Blocked primer molecule (334) comprises a cleavable region (335) and acomplement to the PBD (332) on the circular template molecule (352).

Upon addition of a sample comprising a target nucleic acid of interest(304) (capable of complexing with the gRNA in RNP1 (301)), the targetnucleic acid of interest (304) binds to and activates RNP1 (305) butdoes not interact with or activate RNP2 (302). Once activated, RNP1 cutsthe target nucleic acid of interest (304) via sequence specificcis-cleavage, which activates non-specific trans-cleavage of othernucleic acids present in the reaction mix, including at least one of theblocked primer molecules (334). The circular blocked primer molecule(334), upon cleavage, becomes an unblocked linear primer molecule (344),which has a region (336) complementary to the PBD (332) on the circulartemplate molecule (352) and can hybridize with the circular templatemolecule (352).

Once the unblocked linear primer molecule (344) and the circulartemplate molecule (352) are hybridized (i.e., hybridized at the PBD(332) of the circular template molecule (352) and the PBD complement(336) on the unblocked linear primer molecule (344)), 3′→5′ exonucleaseactivity of the polymerase (338) removes the unhybridizedsingle-stranded DNA at the 3′ end of the unblocked primer molecule(344). The polymerase (338) can now use the circular template molecule(352) (non-target strand) to produce concatenated activating nucleicacid molecules (360) (which are concatenated target strands), which willbe cleaved by the trans-cleavage activity of activated RNP1. The cleavedregions of the concatenated synthesized activating molecules (360)(target strand) are capable of activating the RNP2 (302→308) complex.

As described above, because the nucleic acid-guided nuclease in RNP2(308) comprises both cis- and trans-cleavage activity, more blockedprimer molecules (334) become unblocked primer molecules (344)triggering activation of more RNP2s (308) and more trans-cleavageactivity in a cascade. FIG. 3B at bottom depicts the concurrentactivation of reporter moieties. Intact reporter moieties (309) comprisea quencher (310) and a fluorophore (311). As described above in relationto FIG. 1B, the reporter moieties are also subject to trans-cleavage byactivated RNP1 (305) and RNP2 (308). The intact reporter moieties (309)become activated reporter moieties (312) when the quencher (310) isseparated from the fluorophore (311), and the fluorescent signal (313)is unquenched and can be detected. Signal strength increases rapidly asmore blocked primer molecules (334) become unblocked primer molecules(344) generating synthesized activating nucleic acid molecules andtriggering activation of more RNP2s (308) and more trans-cleavageactivity of the reporter moieties (309). Again, here the reportermoieties are shown as separate molecules from the blocked nucleic acidmolecules, but other configurations may be employed and are discussed inrelation to FIG. 4 . Also note that as with the other embodiments of thecascade assay, in this embodiment, with the exception of the gRNA inRNP1, the cascade assay components stay the same no matter what targetnucleic acid(s) of interest are being detected.

The polymerases used in the “blocked primer molecule” embodiments serveto polymerize a reverse complement strand of the template molecule(non-target strand) to generate a synthesized activating molecule(target strand) as described above. In some embodiments, the polymeraseis a DNA polymerase, such as a BST, T4, or Therminator polymerase (NewEngland BioLabs Inc., Ipswich MA., USA). In some embodiments, thepolymerase is a Klenow fragment of a DNA polymerase. In some embodimentsthe polymerase is a DNA polymerase with 5′→3′ DNA polymerase activityand 3′→5′ exonuclease activity, such as a Type I, Type II, or Type IIIDNA polymerase. In some embodiments, the DNA polymerase, including thePhi29, T7, Q5®, Q5U®, Phusion®, OneTaq®, LongAmp®, Vent®, or Deep Vent®DNA polymerases (New England BioLabs Inc., Ipswich MA., USA), or anyactive portion or variant thereof. Also, a 3′ to 5′ exonuclease can beseparately used if the polymerase lacks this activity.

FIG. 4 depicts three mechanisms in which a cascade assay reaction canrelease a signal from a reporter moiety. FIG. 4 at top shows themechanism discussed in relation to FIGS. 2A, 3A and 3B. In thisembodiment, a reporter moiety 409 is a separate molecule from theblocked nucleic acid molecules present in the reaction mix. Reportermoiety (409) comprises a quencher (410) and a fluorophore (411). Anactivated reporter moiety (412) emits a signal from the fluorophore(411) once it has been physically separated from the quencher (410).

Reporter Moiety Configurations

FIG. 4 at center shows a blocked nucleic acid molecule (403), which isalso a reporter moiety. In addition to quencher (410) and fluorophore(411), a blocking moiety (407) can be seen (see also blocked nucleicacid molecules 203 in FIG. 2A). Blocked nucleic acid molecule/reportermoiety (403) comprises a quencher (410) and a fluorophore (411). In thisembodiment of the cascade assay, when the blocked nucleic acid molecule(403) is unblocked due to trans-cleavage initiated by the target nucleicacid of interest binding to RNP1, the unblocked nucleic acid molecule(406) also becomes an activated reporter moiety with fluorophore (411)separated from quencher (410). Note both the blocking moiety (407) andthe quencher (410) are removed. In this embodiment, reporter signal isdirectly generated as the blocked nucleic acid molecules becomeunblocked. Embodiments of this schema can be used to supply the bulkymodifications to the blocked nucleic acid molecules described below.

FIG. 4 at the bottom shows that cis-cleavage of an unblocked nucleicacid molecule or a synthesized activating molecule at a PAM distalsequence by RNP2 generates a signal. Shown are activated RNP2 (408),unblocked nucleic acid molecule (461), quencher (410), and fluorophore(411) forming an activated RNP2 with the unblocked nucleic acid/reportermoiety intact (460). Cis-cleavage of the unblocked nucleic acid/reportermoiety (461) results in an activated RNP2 with the reporter moietyactivated (462), comprising the activated RNP2 (408), the unblockednucleic acid molecule with the reporter moiety activated (463), quencher(410) and fluorophore (411). Embodiments of this schema also can be usedto supply the bulky modifications to the blocked nucleic acid moleculesdescribed below, and in fact a combination of the configurations ofreporter moieties shown in FIG. 4 at center and at bottom may be used.

Preventing Undesired Blocked Nucleic Acid Molecule Unwinding

The present disclosure improves upon the signal cascade assay describedin U.S. Ser. Nos. 17/861,207; 17/861,208; and 17/861,209 by addressingthe problem with undesired “unwinding” of the blocked nucleic acidmolecule. As described above in detail in relation to FIGS. 1B, 2A, 2B,2G, 3A, 3B, and 4 , the cascade assay is initiated when a target nucleicacid of interest binds to and activates a first pre-assembledribonucleoprotein complex (RNP1). The gRNA of RNP1 (gRNA1), comprising asequence complementary to the target nucleic acid of interest, guidesRNP1 to the target nucleic acid of interest. Upon binding of the targetnucleic acid of interest to RNP1, RNP1 becomes activated, and the targetnucleic acid of interest is cleaved in a sequence specific manner (i.e.,cis-cleavage) while also triggering non-sequence specific,indiscriminate trans-cleavage activity which unblocks the blockednucleic acid molecules in the reaction mix. The unblocked nucleic acidmolecules can then activate a second pre-assembled ribonucleoproteincomplex (RNP2), where RNP2 comprises a second gRNA (gRNA2) comprising asequence complementary to the unblocked nucleic acid molecules, and atleast one of the unblocked nucleic acid molecules is cis-cleaved in asequence specific manner. Binding of the unblocked nucleic acid moleculeto RNP2 leads to cis-cleavage of the unblocked nucleic acid molecule andnon-sequence specific, indiscriminate trans-cleavage activity by RNP2,which in turn unblocks more blocked nucleic acid molecules (and reportermoieties) in the reaction mix activating more RNP2s. Each newlyactivated RNP2 activates more RNP2s, which in turn cleave more blockednucleic acid molecules and reporter moieties in a reaction cascade,where all or most of the signal generated comes from the trans-cleavageactivity of RNP2.

The improvement to the signal boost cascade assay described herein isdrawn to preventing undesired unwinding of the blocked nucleic acidmolecules in the reaction mix before the blocked nucleic acid moleculesare unblocked via trans-cleavage; that is, preventing undesiredunwinding that happens not as a result of unblocking due totrans-cleavage subsequent to cis-cleavage of the target nucleic acid ofinterest or trans-cleavage of unblocked nucleic acid molecules, but dueto other factors. For a description of undesired unwinding, please seeFIG. 1C and the attendant description herein. Minimizing undesiredunwinding serves two purposes. First, preventing undesired unwindingthat happens not as a result of designed or engineered unblocking leadsto a “leaky” cascade assay system, which in turn leads to non-specificsignal generation and false positives.

Second, preventing undesired unwinding limits non-specific interactionsbetween the nucleic acid-guided nucleases (here, the RNP2s) and blockednucleic acid molecules (i.e., the target nucleic acids for RNP2) suchthat only blocked nucleic acid molecules that become unblocked due totrans-cleavage activity react with the nucleic acid-guided nucleases.This “fidelity” in the cascade assay leads primarily to desiredinteractions and limits “wasteful” interactions where the nucleicacid-guided nucleases are essentially interacting with blocked nucleicacid molecules rather than interacting with unblocked nucleic acidmolecules. That is, if unwinding is minimized the nucleic acid-guidednucleases are focused on desired interactions which then leads toimmediate signal generation in the cascade assay. Preventing undesiredunwinding leads to a more efficient cascade assay system providing moreaccurate quantification yet with the rapid results characteristic of thecascade assay (see FIGS. 10A-10H and 12 below).

Ratio of RNP2 to Blocked Nucleic Acid Molecules or Blocked Primers

In one modality to prevent undesired unwinding, the present disclosuredescribes using an unconventional ratio of blocked nucleic acid molecule(i.e., the target molecule for RNP2) and an RNP complex, here RNP2. Theunconventional ratio may be used along with the blocked nucleic acidmolecules and RNP2s described above as a primary method for minimizingunwinding or may be used in combination with the other modalitiesdescribed below to minimize unwinding even more. For example, if onewere to design an ideal blocked nucleic acid molecule having an“infinite K_(d)” such as, e.g., through design of the blocked nucleicacid molecule (or blocked primer molecule) and/or inclusion of bulkymodifications on the blocked nucleic acid molecule (or blocked primermolecule), the ratio of blocked nucleic acid molecules to RNP2s wouldnot affect the reaction mix to any discernable degree. The common wisdomof the ratio of enzyme to target (here, RNP2 to blocked nucleic acidmolecule) is that results are achieved—a signal is generated—when thereis a high concentration of nucleic acid-guided nuclease (i.e., RNPcomplex) and a lower concentration of target or, stated another way,when there is a significant excess of nucleic acid-guided nuclease totarget. As described above, in CRISPR detection/diagnostic assayprotocols known to date, the CRISPR enzyme (i.e., nucleic acid-guidednuclease) is far in excess of blocked nucleic acid molecules (see, Sun,et al., J. of Translational Medicine, 12:74 (2021); Broughton, et al.,Nat. Biotech., 38:870-74 (2020); and Lee, et al., PNAS, 117(41):25722-31(2020)). However, in a cascade assay system where the nucleicacid-guided nuclease (or RNP complex) is in excess of the targets (here,the blocked nucleic acid molecules), the nucleic acid-guided nucleasesencounter the blocked nucleic acid molecules repeatedly, probing theblocked nucleic acid molecules and subjecting them to unwinding. If theblocked nucleic acid molecules are probed and unwound repeatedly, theyfinally unwind which then triggers activation of RNP2 and cis-cleavageof the blocked nucleic acid molecule even in the absence of a targetnucleic acid of interest and the trans-cleavage activity generatedthereby.

However, by adjusting the ratio of RNP2 to blocked nucleic acidmolecules such that there is an excess of blocked nucleic acid moleculesto RNP2, any one blocked nucleic acid molecule may be probed by RNP2;however, the likelihood that any one blocked nucleic acid molecule willbe probed repeatedly (and thus unwound) is much lower. If a blockednucleic acid molecule is probed but then has time to re-hybridize or“recover”, that blocked nucleic acid molecule will stay blocked, willnot be subject to non-specific unwinding, and will not triggeractivation of RNP2. That is, how often any one blocked nucleic acidmolecule is probed is important. As long as an improperly probed blockednucleic acid has time to re-hybridize after unwinding, there is far lesschance that the blocked nucleic acid will be unblocked (i.e., unwound)and will trigger signal generation. That is, preventing non-specificunwinding of the blocked nucleic acid molecules makes the nucleicacid-guided nuclease available for desired unwinding interactions.

In order to prevent non-specific unwinding as described herein, theratio of blocked nucleic acid molecules to RNP2 should be about 50:1, orabout 40:1, or about 35:1, or about 30:1, or about 25:1, or about 20:1,or about 15:1, or about 10:1, or about 7.5:1, or about 5:1, or about4:1, or about 3:1, or about 2.5:1, or about 2:1, or about 1.5:1, or atleast where the molar concentration of blocked nucleic acid molecules isequal to or greater than the molar concentration of RNP2s. As notedabove, the signal amplification cascade assay reaction mixture typicallycontains about 1 fM to about 1 mM of a given RNP2, or about 1 pM toabout 500 μM of a given RNP2, or about 10 pM to about 100 μM of a givenRNP2; thus, the signal amplification cascade assay reaction mixturetypically contains about 2.5 fM to about 2.5 mM blocked nucleic acidmolecules, or about 2.5 pM to about 1.25 mM blocked nucleic acidmolecules, or about 25 pM to about 250 μM blocked nucleic acidmolecules. That is, the reaction mixture contains about 6×10⁴ to about6×10¹⁴ RNP2s per microliter (μl) or about 6×10⁶ to about 6×10¹² RNP2sper microliter (μl) and thus about 6×10⁴ to about 6×10¹⁴ RNP2s permicroliter (μl) or about 6×10⁶ to about 6×10¹² blocked nucleic acidmolecules per microliter (μl). Note, the ratios may be used along withthe blocked nucleic acid molecules and RNP2s described above as aprimary method for minimizing unwinding or the ratios of blocked nucleicacid molecules to RNP2s may be used in combination with the othermodalities described below to further minimize unwinding. Again, if onewere to design an ideal blocked nucleic acid molecule having an“infinite K_(d)”, the ratio of blocked nucleic acid molecules to RNP2swould not affect the reaction mix to any discernable degree and theratios of blocked nucleic acid molecules to RNP2s would not necessarilybe within these ranges.

Variant Engineered Nucleic Acid-Guided Nucleases

In some embodiments, the protein sequence of the Cas12a nucleicacid-guided nuclease is modified, with e.g., mutations to the domainsthat interact with the PAM region or surrounding sequences on theblocked nucleic acid molecules (see Shin et al., Front. Genet., 11:1577(2021); doi: 10.3389/fgene.2020.571591, herein incorporated byreference; and Yamano et al., Mol. Cell, 67(4): 633-645 (2017); doi:10.1016/j.molcel.2017.06.035, herein incorporated by reference) suchthat the variant engineered nucleic acid-guided nuclease has reduced (orabsent) PAM specificity, relative to the unmodified or wildtype nucleicacid-guided nuclease and reduced cleavage activity in relation to doublestrand DNA with or without a PAM. Such enzymes are referred to herein assingle-strand-specific Cas12a nucleic acid-guided nucleases or variantengineered nucleic acid-guided nucleases.

FIG. 5 is a simplified block diagram of an exemplary method 500 fordesigning, synthesizing and screening variant nucleic acid-guidednucleases. In a first step, mutations or modifications to a nucleicacid-guided nuclease are designed 502, based on, e.g., homology torelated nucleic acid-guided nucleases, predicted protein structure andactive site configuration, and mutagenesis modeling. For assessment ofhomologies to other nucleic acid-guided nucleases, amino acid sequencesmay be found in publicly available databases known to those with skillin the art, including, e.g., Protein DataBank Europe (PDBe), ProteinDatabank Japan (PDBj), SWISS-PROT, GenBank, RefSeq, TrEMBL, PROSITE,DisProt, InterPro, PIR-International, and PRF/SEQDB. Amino acid homologyalignments for purposes of determining similarities to known nucleicacid-guided nucleases can be performed using CUSTALW, CUSTAL OMEGA,COBALT: Multiple Alignment Tool; SIM; and PROBCONS.

For protein engineering and amino acid substitution model predictionsfor each of the desired mutations, protein modeling software such asSWISS-MODEL, HHpred, I-TASSER, IntFOLD, RaptorX, FoldX, Rosetta, andtrRosetta may be used to simulate the structural change(s) and tocalculate various parameters due to the structural changes as a resultof the amino acid substitution(s), including root mean square deviation(RMSD) value in Angstrom units (i.e., a measurement of the differencebetween the backbones of the initial nucleic acid-guided nuclease andthe mutated nucleic acid nucleic acid-guided nuclease) and changes tothe number of hydrogen bonds and conformation in the active site. Forthe methods used to generate the variant engineered nucleic acid-guidednucleases described herein, see Example VII below.

Following modelling, coding sequences for the variant nucleicacid-guided nucleases that appear to deliver desired properties aresynthesized and inserted into an expression vector 504. Methods forsite-directed mutagenesis are known in the art, including PCR-basedmethods such as traditional PCR, where primers are designed to includethe desired change; primer extension, involving incorporating mutagenicprimers in independent nested PCR before combining them in the finalproduct; and inverse PCR. Additionally, CRISPR gene editing may beperformed to introduce the desired mutation or modification to thenucleic acid-guided nuclease coding sequence. The mutated (variant)coding sequences are inserted into an expression vector backbonecomprising regulatory sequences such as enhancer and promoter regions.The type of expression vector (e.g., plasmid or viral vector) will varydepending on the type of cells to be transformed.

At step 506, cells of choice are transformed with the variant expressionvectors. A variety of delivery systems may be used to introduce (e.g.,transform or transfect) the expression vectors into a host cell,including the use of yeast systems, lipofection systems, microinjectionsystems, biolistic systems, virosomes, liposomes, immunoliposomes,polycations, lipid:nucleic acid conjugates, virions, artificial virions,viral vectors, electroporation, cell permeable peptides, nanoparticles,nanowires, exosomes. Once cells are transformed (or transfected), thetransformants are allowed to recover and grow.

Following transformation, the cells are screened for expression ofnucleic acid-guided nucleases with desired properties 508, such as cutactivity or lack thereof, paste activity or lack thereof, PAMrecognition or changes thereto, stability and the ability to form RNPsat various temperatures, and/or cis- and trans-cleavage activity atvarious temperatures. The assays used to screen the variant nucleicacid-guided nucleases will vary depending on the desired properties, butmay include in vitro and in vivo PAM depletion, assays for editingefficiency such as a GFP to BFP assay, and, as used to assess thevariant nucleic acid-guided nucleases described herein, in vitrotranscription/translation (IVTT) assays were used to measure in vitrotrans cleavage with both dsDNA and ssDNA and with and without thepresence of a PAM in the blocked nucleic acid molecules, where dsDNAshould not activate trans-cleavage regardless of the presence of PAMsequence.

After screening the variant nucleic acid-guided nucleases via the IVTTassays, variants with the preferred properties are identified andselected 510. At this point, a variant may be chosen 512 to go forwardinto production for use in, e.g., the CRISPR cascade systems describedherein; alternatively, promising mutations and/or modifications may becombined 514 and the construction, screening and identifying process isrepeated.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease may not recognize one or more of the following PAMor partial PAM sequences (listed from 5′ to 3′): TTTN, TTTV, CTTA, CTTV,TCTV, TTCV, YTV, or YTN wherein “A” represents adenine, “C” representscytosine, “T” represents thymine, “G” represents guanine, “V” representsguanine or cytosine or adenine, “Y” represents guanine or adenine, and“N” represents any nucleotide. In some embodiments, the Cas12a nucleicacid-guided nuclease may have reduced recognition for one or more of thefollowing PAM or partial PAM sequences (listed from 5′ to 3′): TTTN,TTTV, CTTA, CTTV, TCTV, TTCV, YTV, or YTN. The single-strand-specificCas12a nucleic acid-guided nucleases described herein may have at least50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or 100%, such as about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 100%) reduced recognition (i.e., specificity) forone or more of the following PAM or partial PAM sequences (listed from5′ to 3′): TTTN, TTTV, CTTA, CTTV, TCTV, TTCV, YTV, or YTN.

Exemplary wild type (WT) Cas12a protein sequences are described in Table7 below. FIG. 6A shows the result of protein structure prediction usingRosetta and SWISS modeling of wildtype LbCas12a (Lachnospriaceaebacterium Cas12a), and FIG. 6B shows the result of example mutations onthe LbCas12a protein structure prediction using Rosetta and SWISSmodeling of LbCas12a and indicating the PAM regions (described in moredetail in relation to Example VII). Any of these sequences (e.g., SEQ IDNOs: 1-15 and homologs or orthologs thereof) may be modified, asdescribed herein, to generate a single-strand-specific nucleicacid-guided nuclease.

TABLE 7 Exemplary wild type Cas12a nucleic acid-guided nucleases SpeciesSEQ Name ID Reference ID NO: Protein Sequence Lachnospiraceae SEQMSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAED bacterium Cas12a IDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENK (LbCas12a) NO: 1ELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIAL PDD: 6KL9_AVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH Acidaminococcus SEQMTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH sp. Cas12a IDYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETR (AsCas12a) NO: 2NALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFN NCBI Ref.:GKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDI WP_021736722.1STAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISN QDWLAYIQELRN Candidatus SEQMNNYDEFTKLYPIQKTIRFELKPQGRTMEHLETFNFFEEDRDRAEK Methanoplasma IDYKILKEAIDEYHKKFIDEHLTNMSLDWNSLKQISEKYYKSREEKDK termitum NO: 3KVFLSEQKRMRQEIVSEFKKDDRFKDLFSKKLFSELLKEEIYKKGN (CtCas12a)HQEIDALKSFDKFSGYFIGLHENRKNMYSDGDEITAISNRIVNENFP NCBI Gene ID:KFLDNLQKYQEARKKYPEWIIKAESALVAHNIKMDEVFSLEYFNK 24818655VLNQEGIQRYNLALGGYVTKSGEKMMGLNDALNLAHQSEKSSKGRIHMTPLFKQILSEKESFSYIPDVFTEDSQLLPSIGGFFAQIENDKDGNIFDRALELISSYAEYDTERIYIRQADINRVSNVIFGEWGTLGGLMREYKADSINDINLERTCKKVDKWLDSKEFALSDVLEAIKRTGNNDAFNEYISKMRTAREKIDAARKEMKFISEKISGDEESIHIIKTLLDSVQQFLHFFNLFKARQDIPLDGAFYAEFDEVHSKLFAIVPLYNKVRNYLTKNNLNTKKIKLNFKNPTLANGWDQNKVYDYASLIFLRDGNYYLGIINPKRKKNIKFEQGSGNGPFYRKMVYKQIPGPNKNLPRVFLTSTKGKKEYKPSKEIIEGYEADKHIRGDKFDLDFCHKLIDFFKESIEKHKDWSKFNFYFSPTESYGDISEFYLDVEKQGYRMHFENISAETIDEYVEKGDLFLFQIYNKDFVKAATGKKDMHTIYWNAAFSPENLQDVVVKLNGEAELFYRDKSDIKEIVHREGEILVNRTYNGRTPVPDKIHKKLTDYHNGRTKDLGEAKEYLDKVRYFKAHYDITKDRRYLNDKIYFHVPLTLNFKANGKKNLNKMVIEKFLSDEKAHIIGIDRGERNLLYYSIIDRSGKIIDQQSLNVIDGFDYREKLNQREIEMKDARQSWNAIGKIKDLKEGYLSKAVHEITKMAIQYNAIVVMEELNYGFKRGRFKVEKQIYQKFENMLIDKMNYLVFKDAPDESPGGVLNAYQLTNPLESFAKLGKQTGILFYVPAAYTSKIDPTTGFVNLFNTSSKTNAQERKEFLQKFESISYSAKDGGIFAFAFDYRKFGTSKTDHKNVWTAYTNGERMRYIKEKKRNELFDPSKEIKEALTSSGIKYDGGQNILPDILRSNNNGLIYTMYSSFIAAIQMRVYDGKEDYIISPIKNSKGEFFRTDPKRRELPIDADANGAYNIALRGELTMRAIAEKFDPDSEKMAKLELKHKDWFEFMQTRGD Eubacterium SEQMNGNRSIVYREFVGVIPVAKTLRNELRPVGHTQEHIIQNGLIQEDEL eligens IDRQEKSTELKNIMDDYYREYIDKSLSGVTDLDFTLLFELMNLVQSSP (EeCas12a) NO: 4SKDNKKALEKEQSKMREQICTHLQSDSNYKNIFNAKLLKEILPDFI NCBI Gene ID:KNYNQYDVKDKAGKLETLALFNGFSTYFTDFFEKRKNVFTKEAVS 41356122TSIAYRIVHENSLIFLANMTSYKKISEKALDEIEVIEKNNQDKMGDWELNQIFNPDFYNMVLIQSGIDFYNEICGVVNAHMNLYCQQTKNNYNLFKMRKLHKQILAYTSTSFEVPKMFEDDMSVYNAVNAFIDETEKGNIIGKLKDIVNKYDELDEKRIYISKDFYETLSCFMSGNWNLITGCVENFYDENIHAKGKSKEEKVKKAVKEDKYKSINDVNDLVEKYIDEKERNEFKNSNAKQYIREISNIITDTETAHLEYDDHISLIESEEKADEMKKRLDMYMNMYHWAKAFIVDEVLDRDEMFYSDIDDIYNILENIVPLYNRVRNYVTQKPYNSKKIKLNFQSPTLANGWSQSKEFDNNAIILIRDNKYYLAIFNAKNKPDKKIIQGNSDKKNDNDYKKMVYNLLPGANKMLPKVFLSKKGIETFKPSDYIISGYNAHKHIKTSENFDISFCRDLIDYFKNSIEKHAEWRKYEFKFSATDSYSDISEFYREVEMQGYRIDWTYISEADINKLDEEGKIYLFQIYNKDFAENSTGKENLHTMYFKNIFSEENLKDIIIKLNGQAELFYRRASVKNPVKHKKDSVLVNKTYKNQLDNGDVVRIPIPDDIYNEIYKMYNGYIKESDLSEAAKEYLDKVEVRTAQKDIVKDYRYTVDKYFIHTPITINYKVTARNNVNDMVVKYIAQNDDIHVIGIDRGERNLIYISVIDSHGNIVKQKSYNILNNYDYKKKLVEKEKTREYARKNWKSIGNIKELKEGYISGVVHEIAMLIVEYNAIIAMEDLNYGFKRGRFKVERQVYQKFESMLINKLNYFASKEKSVDEPGGLLKGYQLTYVPDNIKNLGKQCGVIFYVPAAFTSKIDPSTGFISAFNFKSISTNASRKQFFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITMGKTQWTVYTNGERLQSEFNNARRTGKTKSINLTETIKLLLEDNEINYADGHDIRIDMEKMDEDKKSEFFAQLLSLYKLTVQMRNSYTEAEEQENGISYDKIISPVINDEGEFFDSDNYKESDDKECKMPKDADANGAYCIALKGLYEVLKIKSEWTEDGFDRNCLKLPHAEWLDFIQNKRYE Moraxella SEQMLFQDFTHLYPLSKTVRFELKPIGKTLEHIHAKNFLNQDETMADM bovoculi Cas12a IDYQKVKAILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKD (Mb3Cas12a) NO: 5DGLQKQLKDLQAVLRKEIVKPIGNGGKYKAGYDRLFGAKLFKDG GenBank:KELGDLAKFVIAQEGESSPKLAHLAHFEKFSTYFTGFHDNRKNMY AKG12737.1SDEDKHTAIAYRLIHENLPRFIDNLQILATIKQKHSALYDQIINELTASGLDVSLASHLDGYHKLLTQEGITAYNTLLGGISGEAGSRKIQGINELINSHHNQHCHKSERIAKLRPLHKQILSDGMGVSFLPSKFADDSEVCQAVNEFYRHYADVFAKVQSLFDGFDDYQKDGIYVEYKNLNELSKQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFIKGVHSLASLEQAIEHYTARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNNHSTIKGFLERERPAGERALPKIKSDKSPEIRQLKELLDNALNVAHFAKLLTTKTTLHNQDGNFYGEFGALYDELAKIATLYNKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQKDGCYYLALLDKAHKKVFDNAPNTGKSVYQKMIYKLLPGPNKMLPKVFFAKSNLDYYNPSAELLDKYAQGTHKKGDNFNLKDCHALIDFFKAGINKHPEWQHFGFKFSPTSSYQDLSDFYREVEPQGYQVKFVDINADYINELVEQGQLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDNLVNPIYKLNGEAEIFYRKASLDMNETTIHRAGEVLENKNPDNPKKRQFVYDIIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIQQYDEVNVIGIDRGERHLLYLTVINSKGEILEQRSLNDITTASANGTQMTTPYHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQISQLMLKYNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHLVLKDKADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTSKIDPETGFVDLLKPRYENIAQSQAFFGKFDKICYNADRGYFEFHIDYAKFNDKAKNSRQIWKICSHGDKRYVYDKTANQNKGATIGVNVNDELKSLFTRYHINDKQPNLVMDICQNNDKEFHKSLMYLLKTLLALRYSNASSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGLWLLNELKNSDDLNKVKLAIDNQTWLNFAQNR Francisella SEQMSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDY novicida Cas12a IDKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNL (FnCas12a) NO: 6QKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLIL UniProtKB/Swiss-WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRK Prot: A0Q7Q2.1NVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVEDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQ NRNN Francisella SEQMSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDY tularensis subsp. IDKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNL novicida FTG NO: 7QKDFKSAKDTIKKQISKYINDSEKFKNLFNONLIDAKKGQESDLIL Cas12aWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRK (FnoCas12a)NVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIK NCBI Gene ID:KDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFN 60806594TIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVEDDYSVIGTAVLEYITQQVAPKNLDNPSKKEQDLIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILSNFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEEDVKAIKDLLDQTNNLLHRLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLASGWDKNKESANTAILFIKDDKYYLGIMDKKHNKIFSDKAIEENKGEGYKKIVYKQIADASKDIQNLMIIDGKTVCKKGRKDRNGVNRQLLSLKRKHLPENIYRIKETKSYLKNEARFSRKDLYDFIDYYKDRLDYYDFEFELKPSNEYSDFNDFTNHIGSQGYKLTFENISQDYINSLVNEGKLYLFQIYSKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKETIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDNFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLDRIKNNQEGKKLNLVIK NEEYFEFVQNRNNFlavobacteriales SEQ MKNNNMLNFTNKYQLSKTLRFELKPIGKTKENIIAKNILKKDEERAbacterium ID ESYQLMKKTIDGFHKHFIELAMQEVQKTKLSELEEFAELYNKSAEE (FbCas12a)NO: 8 KKKDDKFDDKFKKVQEALRKEIVKGFNSEKVKYYYSNIDKKILFT NCBI Gene ID:ELLKNWIPNEKMITELSEWNAKTKEEKEHLVYLDKEFENFTTYFG MBE7442138.1GFHKNRENMYTDKEQSTAIAYRLIHENLPKFLDNINIYKKVKEIPVLREECKVLYKEIEEYLNVNSIDEVFELSYYNKTLTQKDIDVYNLIIGGRTLEEGKKKIQGLNEYINLYNQKQEKKNRIPKLKILYKQILSDRDSISWLPESFEDDNEKTASQKVLEAINLYYRDNLLCFQPKDKKDTENVLEETKKLLAGLSTSDLSKIYIRNDRAITDISQALFKDYGVIKDALKFQFIQSFTIGKNGLSKKQEEAIEKHLKQKYFSIAEIENALFTYQSETDALKELKENSHPVVDYFINHFKAKKKEETDKDFDLIANIDAKYSCIKGLLNTPYPKDKKLYQRSKGDNDIDNIKAFLDALMELLHFVKPLALSNDSTLEKDQNFYSHFEPYYEQLELLIPLYNKVRNFAAKKPYSTEKFKLNFDNATLLNGWDKNKETDNTSVILRKDGLYYLAIMPQDNKNVFKDSPDLKANENCFEKMDYKQMALPMGFGAFVRKCFGTASQLGWNCPESCKNEEDKIIIKEDEVKNNRAEIIDCYKDFLNIYEKDGFQYKEYGFDFKESNKYESLREFFIDVEQQGYKITFQNISENYINQLVEDGKLYLFQIYNKDFSPYSKGKPNMHTMYWKALFDSENLKDVVYKLNGQAEVFYRKKSIEQKNIVTHKANEPIDNKNPKAKKKQSTFEYDLIKDKRYTVDKFQFHVPITLNFKATGNDYINQDVLTYLKNNPEVNIIGLDRGERHLIYLTLINQKGEILLQESLNTIVNKKYDIETPYHTLLQNKEDERAKARENWGVIENIKELKEGYISQVVHKIAKLMVEYNAIVVMEDLNTGFKRGRFKVEKQVYQKLEKMLIDKLNYLVFKDKDPSEVGGLYHALQLTNKFENFSKIGKQSGFLFYVPAWNTSKIDPTTGFVNLFNTKYESVPKAQEFFKKFKSIKFNSAENYFEFAFDYNDFTTRAEGTKTDWIVCTYGDRIKTFRNPDKVNQWDNQEVNLTEQFEDFFGKNNLIYGDGNCIKNQIILHDKKEFFEGLLHLLKLTLQMRNSITNSEVDYLISPVKNNKGEFYDSRKANNTLPKDADANGAYHIAKKGLVLLNRLKENEVEEFEKSKKVKDGKSQWLPNKDWLDFVQRNVEDMVVV Lachnospira SEQMNGNRSIVYREFVGVTPVAKTLRNELRPVGHTQEHIIQNGLIQEDE eligens IDLRQEKSTELKNIMDDYYREYIDKSLSGVTDLDFTLLFELMNLVQSS (Lb4Cas12a) NO: 9PSKDNKKALEKEQSKMREQICTHLQSDSNYKNIFNAKLFKEILPDFI NCBI Gene ID:KNYNQYDVKDKAGKLETVALFNGFSTYFTDFFEKRKNVFTKEAV MBS6299380.1STSIAYRIVHENSLIFLANMTSYKKISEKALDEIEVIEKNNQDKMGDWELNQIFNPDFYNMVLIQSGIDFYNEICGVVNAHMNLYCQQTRNNYNLFKMRKLHKQILAYTSTSFEVPKMFEDDMSVYNAVNAFIDETEKGNIIVKLKDIVNKYDELDEKRIYISKDFYETLSCFISGNWNLITGCVENFYDENIHAKGKSKEEKVKKAVKEDKYKSINDVNDLVEKYIDEKERNEFKNSNAKQYIREISNIITDTETAHLEYDEHISLIESEEKADEMKKRLDMYMNMYHWAKAFIVDEVLDRDEMFYSDIDDIYNILENIVPLYNRVRNYVTQKPYNSKKIKLNFQSPTLANGWSQSKEFDNNAIILIRDNKYYLAIFNAKNKPDKKIIQGNSDKKNDNDYKKMVYNLLPGANKMLPKVFLSKKGIETFKPSDYIISGYNAHKHIKTSENFDISFCRDLIDYFKNSIEKHAEWRKYEFKFSATDSYNDISEFYREVEMQGYRIDWTYISEADINKLDEEGKIYLFQIYNKYFAENSTGKENLHTMYFKNIFSEENLKDIIIKLNGQAELFYRRASVKNPVKHKKDSVLVNKTYKNQLDNGDVVRIPIPDDIYNEIYKMYNGYIKESDLSEAAKEYLDKVEVRTAQKDIVKDYRYTVDKYFIHTPITINYKVTARNNVNDMAVKYIAQNDDIHVIGIDRGERNLIYISVIDSHGNIVKQKSYNILNNYDYKKKLVEKEKTREYARKNWKSIGNIKELKEGYISGVVHEIAMLMVEYNAIIAMEDLNYGFKRGRFKVERQVYQKFESMLINKLNYFASKGKSVDEPGGLLRGYQLTYVPDNIKNLGKQCGVIFYVPAAFTSKIDPSTGFISAFNFKSISTNASRKQFFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITMGKTQWTVYTNGERLQSEFNNARRTGKTKSINLTETIKLLLKDNKINYADGHDVRIDMEKMDEDKNSEFFAQLLSLYKLTVQMRNSYTEAEEQEKGISYDKIISPVINDEGEFFDSDNYKESDDKECKMPKDADANGAYCIALKGLYEVLKIKSEWTEDGFDRNCLKLPHAEWLDFIQNKRY E Moraxella SEQMLFQDFTHLYPLSKTVRFELKPIGRTLEHIHAKNFLSQDETMADMY bovoculi IDQKVKVILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKDD (MbCas12a) NO:GLQKQLKDLQAVLRKESVKPIGSGGKYKTGYDRLFGAKLFKDGK NCBI Gene ID: 10ELGDLAKFVIAQEGESSPKLAHLAHFEKFSTYFTGFHDNRKNMYS WP_046697655.1DEDKHTAIAYRLIHENLPRFIDNLQILTTIKQKHSALYDQIINELTASGLDVSLASHLDGYHKLLTQEGITAYNRIIGEVNGYTNKHNQICHKSERIAKLRPLHKQILSDGMGVSFLPSKFADDSEMCQAVNEFYRHYTDVFAKVQSLFDGFDDHQKDGIYVEHKNLNELSKQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFIKGVHSLASLEQAIEHHTARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNNHSTIKGFLERERPAGERALPKIKSGKNPEMTQLRQLKELLDNALNVAHFAKLLTTKTTLDNQDGNFYGEFGVLYDELAKIPTLYNKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQKDGCYYLALLDKAHKKVFDNAPNTGKNVYQKMVYKLLPGPNKMLPKVFFAKSNLDYYNPSAELLDKYAKGTHKKGDNFNLKDCHALIDFFKAGINKHPEWQHFGFKFSPTSSYRDLSDFYREVEPQGYQVKFVDINADYIDELVEQGKLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDNLADPIYKLNGEAQIFYRKASLDMNETTIHRAGEVLENKNPDNPKKRQFVYDIIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIQQYDEVNVIGIDRGERHLLYLTVINSKGEILEQRSLNDITTASANGTQVTTPYHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQINQLMLKYNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHLVLKDKADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTSKIDPETGFVDLLKPRYENIAQSQAFFGKFDKICYNTDKGYFEFHIDYAKFTDKAKNSRQKWAICSHGDKRYVYDKTANQNKGAAKGINVNDELKSLFARYHINDKQPNLVMDICQNNDKEFHKSLMCLLKTLLALRYSNASSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGLWLLNELKNSDDLNKVKLAIDNQTWLNFAQNR Prevotella bryantii SEQMKFTDFTGLYSLSKTLRFELKPIGKTLENIKKAGLLEQDQHRADSY (Pb2Cas12a) IDKKVKKIIDEYHKAFIEKSLSNFELKYQSEDKLDSLEEYLMYYSMKR NCBI Gene ID: NO:IEKTEKDKFAKIQDNLRKQIADHLKGDESYKTIFSKDLIRKNLPDFV WP_039871282.1 11KSDEERTLIKEFKDFTTYFKGFYENRENMYSAEDKSTAISHRIIHENLPKFVDNINAFSKIILIPELREKLNQIYQDFEEYLNVESIDEIFHLDYFSMVMTQKQIEVYNAIIGGKSTNDKKIQGLNEYINLYNQKHKDCKLPKLKLLFKQILSDRIAISWLPDNFKDDQEALDSIDTCYKNLLNDGNVLGEGNLKLLLENIDTYNLKGIFIRNDLQLTDISQKMYASWNVIQDAVILDLKKQVSRKKKESAEDYNDRLKKLYTSQESFSIQYLNDCLRAYGKTENIQDYFAKLGAVNNEHEQTINLFAQVRNAYTSVQAILTTPYPENANLAQDKETVALIKNLLDSLKRLQRFIKPLLGKGDESDKDERFYGDFTPLWETLNQITPLYNMVRNYMTRKPYSQEKIKLNFENSTLLGGWDLNKEHDNTAIILRKNGLYYLAIMKKSANKIFDKDKLDNSGDCYEKMVYKLLPGANKMLPKVFFSKSRIDEFKPSENIIENYKKGTHKKGANFNLADCHNLIDFFKSSISKHEDWSKFNFHFSDTSSYEDLSDFYREVEQQGYSISFCDVSVEYINKMVEKGDLYLFQIYNKDFSEFSKGTPNMHTLYWNSLFSKENLNNIIYKLNGQAEIFFRKKSLNYKRPTHPAHQAIKNKNKCNEKKESIFDYDLVKDKRYTVDKFQFHVPITMNFKSTGNTNINQQVIDYLRTEDDTHIIGIDRGERHLLYLVVIDSHGKIVEQFTLNEIVNEYGGNIYRTNYHDLLDTREQNREKARESWQTIENIKELKEGYISQVIHKITDLMQKYHAVVVLEDLNMGFMRGRQKVEKQVYQKFEEMLINKLNYLVNKKADQNSAGGLLHAYQLTSKFESFQKLGKQSGFLFYIPAWNTSKIDPVTGFVNLFDTRYESIDKAKAFFGKFDSIRYNADKDWFEFAFDYNNFTTKAEGTRTNWTICTYGSRIRTFRNQAKNSQWDNEEIDLTKAYKAFFAKHGINIYDNIKEAIAMETEKSFFEDLLHLLKLTLQMRNSITGTTTDYLISPVHDSKGNFYDSRICDNSLPANADANGAYNIARKGLMLIQQIKDSTSSNRFKFSPITNKDWLIFAQEK PYLND Candidatus SEQMENKNNQTQSIWSVFTKKYSLQKTLRFELKPVGETKKWLEENDIF Parcubacteria IDKKDLNIDKSYNQAKFYFDKLHQDFIKESLSVENGIRNIDFEKFAKIF bacterium NO:ESNKEKIVSLKKKNKEVKDKNKKNWDEISKLEKEIEGQRENLYKEI (PgCas12a) 12RELFDKRAEKWKKEYQDKEIERGGKKEKIKFSSADLKQKGVNFLT NCBI Gene ID:AAGIINILKYKFPAEKDEEFRKEGYPSLFINDELNPGKKIYIFESFDK BCX15829.1FTTYLSKFQQTRENLYKDDGTSTAVATRIVSNFERFLENKSLFEEKYKNKAKDVGLTKEEEKVFEINYYYDCLIQEGIDKYNKIIGEINRKTKEYRDKNKIDKKDLPLFLNLEKQILGEVKKERVFIEAKDEKTEEEVFIDRFQEFIKRNKIKIYGDEKEEIEGAKKFIEDFTSGIFENDYQSIYLKKNVINEIVNKWFSNPEEFLMKLTGVKSEEKIKLKKFTSLDEFKNAILSLEGDIFKSRFYKNEVNPEAPLEKEEKSNNWENFLKIWRFEFESLFKDKVEKGEIKKDKNGEPIQIFWGYTDKLEKEAEKIKFYSAEKEQIKTIKNYCDAALRINRMMRYFNLSDKDRKDVPSGLSTEFYRLVDEYFNNFEFNKYYNGIRNFITKKPSDENKIKLNFESRSLLDGWDVSKEKDNLGLIFIKNNKYYLGVLRKENSKLFDYQITEKDNQKEKERKNNLKNEILANDNEDFYLKMNYWQIADPAKDIFNLVLMPDNTVKRFTKLEEKNKHWPDEIKRIKEKGTYKREKVNREDLVKIINYFRKCALIYWKKFDLKLLPSEEYQTFKDFTDHIALQGYKINFDKIKASYIEKQLNDGNLYLFEVSNKDFYKYKKPDSRKNIHTLYWEHIFSKENLEEIKYPLIRLNGKAEIFYRDVLEMNEEMRKPVILERLNGAKQAKREDKPVYHYQRYLKPTYLFHCPITLNADKPSSSFKNFSSKLNHFIKDNLGKINIIGIDRGEKNLLYYCVINQNQEILDYGSLNKINLNKVNNVNYFDKLVEREKQRQLERQSWEPVAKIKDLKQGYISYVVRKICDLIINHNAIVVLEDLSRRFKQIRNGISERTVYQQFEKALIDKLNYLIFKDNRDVFSPGGVLNGYQLAAPFTSFKDIEKAKQTGVLFYTSAEYTSQTDPLTGFRKNIYISNSASQEKIKELINKLKKFGWDDTEESYFIEYNQVDFAEKKKKPLSKDWTIWTKVPRVIRWKESKSSYWSYKKINLNEEFRDLLEKYGFEAQSNDILSNLKKRIAENDKLLVEKKEFDGRLKNFYERFIFLFNIVLQVRNTYSLSVEIDKTEKKLKKIDYGIDFFASPVKPFFTTFGLREIGIEKDGKVVKDNAREEIASENLAEFKDRLKEYKPEEKFDADGVGAYNIARKGLIILEKIKNNPNKPDLSISKEEWDKFVQR Acidaminococcus SEQMTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH sp. IDYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETR (AaCas12a) NO:NALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFN NCBI Gene ID: 13GKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDI WP_021736722.1STAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISN QDWLAYIQELRN BacteroidetesSEQ MESPTTQLKKFTNLYQLSKTLRFELKPVGKTKEHIETKGILKKDEE bacterium IDRAVNYKLIKKIIDGFHKHFIELAMQQVKLSKLDELAELYNASAERK (BoCas12a) NO:KEESYKKELEQVQAALRKEIVKGFNIGEAKEIFSKIDKKELFTELLD NCBI Gene ID: 14EWVKNLEEKKLVDDFKTFTTYFTGFHENRKNMYTDKAQSTAIAY PKP47250.1RLVHENLPKFLDNTKIFKQIETKFEASKIEEIETKLEPIIQGTSLSEIFTLDYYNHALTQAGIDFINNIIGGYTEDEGKKKIQGLNEYINLYNQKQEKKNRIPKLKILYKQILSDRDSISFLPDAFEDSQEVLNAIQNYYQTNLIDFKPKDKEETENVLEETKKLLTELFSNELSKIYIRNDKAITDISQALFNDWGVFKSALEYKFIQDLELGTKELSKKQENEKEKYLKQAYFSIAEIENALFAYQNETDVLNEIKENSHPIADYFTKHFKAKKKVDTSTSSVEKDFDLIANIDAKYSCIKGILNTDYPKDKKLNQEKKTIDDLKVFLDSLMELLHFVKPLALPNDSILEKDENFYSHFESYYEQLELLIPLYNKVRNYAAKKPYSTEKFKLNFENATLLKGWDKNKEIDNTSVILRKRGLYYLAIMPQDNKNVFKKSPNLKNNESCFEKMDYKQMALPMGFGAFVRKCFGTAFQLGWNCPKSCINEEDKIIIKEDEVKNNRAEIIDCYKDFLNIYEKDGFQYKEYGFNFKESKEYESLREFFIDVEQKGYKIEFQNISENYIHQLVNEGKLYLFQIYNKDFSSYSKGKPNMHTMYWKALFDPENLKDVVYKLNGQAEVFYRKKSIEDKNIITHKANEPIENKNPKAKKTQSTFEYDLIKDKRYTVDKFHFHVPITINFKATGNNYINQQVLDHLKNNTDVNIIGLDRGERHLIYLTLINQKGEILLQESLNTIVNKKFDIETPYHTLLQNKEDERAKARENWGVIENIKELKEGYLSQVVHKIAKLMVDYNAIVVMEDLNTGFKRGRFKVEKQVYQKLEKMLIDKLNYLVFKDKDPNEVGGLYNALQLTNKFESFSKMGKQSGFLFYVPAWNTSKIDPTTGFVNLFYAKYESIPKAQDFFTKFKSIRYNSDENYFEFAFDYNDFTTRAEGTKSDWTVCTYGDRIKTFRNPEKNNQWDNQEVNLIEQFEAFFGKHNITYGDGNCIKKQLIEQDKKEFFEELFHLFKLTLQMRNSITNSEIDYLISPVKNSKKEFYDSRKADSTLPKDADANGAYHIAKKGLMWLEKINSFKGSDWKKLDLDKTNKTWLNFVQETASEKHKKLQTV Candidatus SEQMDAKEFTGQYPLSKTLRFELRPIGRTWDNLEASGYLAEDRHRAEC Methanomethylop IDYPRAKELLDDNHRAFLNRVLPQIDMDWHPIAEAFCKVHKNPGNK hilus alvus NO:ELAQDYNLQLSKRRKEISAYLQDADGYKGLFAKPALDEAMKIAKE Mx1201 15NGNESDIEVLEAFNGFSVYFTGYHESRENIYSDEDMVSVAYRITED (CMaCas12a)NFPRFVSNALIFDKLNESHPDIISEVSGNLGVDDIGKYFDVSNYNNF NCBI Gene ID:LSQAGIDDYNHIIGGHTTEDGLIQAFNVVLNLRHQKDPGFEKIQFK 15139718QLYKQILSVRTSKSYIPKQFDNSKEMVDCICDYVSKIEKSETVERALKLVRNISSFDLRGIFVNKKNLRILSNKLIGDWDAIETALMHSSSSENDKKSVYDSAEAFTLDDIFSSVKKFSDASAEDIGNRAEDICRVISETAPFINDLRAVDLDSLNDDGYEAAVSKIRESLEPYMDLFHELEIFSVGDEFPKCAAFYSELEEVSEQLIEIIPLFNKARSFCTRKRYSTDKIKVNLKFPTLADGWDLNKERDNKAAILRKDGKYYLAILDMKKDLSSIRTSDEDESSFEKMEYKLLPSPVKMLPKIFVKSKAAKEKYGLTDRMLECYDKGMHKSGSAFDLGFCHELIDYYKRCIAEYPGWDVFDFKFRETSDYGSMKEFNEDVAGAGYYMSLRKIPCSEVYRLLDEKSIYLFQIYNKDYSENAHGNKNMHTMYWEGLFSPQNLESPVFKLSGGAELFFRKSSIPNDAKTVHPKGSVLVPRNDVNGRRIPDSIYRELTRYFNRGDCRISDEAKSYLDKVKTKKADHDIVKDRRFTVDKMMFHVPIAMNFKAISKPNLNKKVIDGIIDDQDLKIIGIDRGERNLIYVTMVDRKGNILYQDSLNILNGYDYRKALDVREYDNKEARRNWTKVEGIRKMKEGYLSLAVSKLADMIIENNAIIVMEDLNHGFKAGRSKIEKQVYQKFESMLINKLGYMVLKDKSIDQSGGALHGYQLANHVTTLASVGKQCGVIFYIPAAFTSKIDPTTGFADLFALSNVKNVASMREFFSKMKSVIYDKAEGKFAFTFDYLDYNVKSECGRTLWTVYTVGERFTYSRVNREYVRKVPTDIIYDALQKAGISVEGDLRDRIAESDGDTLKSIFYAFKYALDMRVENREEDYIQSPVKNASGEFFCSKNAGKSLPQDSDANGAYNIALKGILQLRMLSEQYDPNAESIRLPLITNKAWLTFMQSGMKTWKN

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with LbCas12a): K538A, K538D, K538E, Y542A, Y542D, Y542E, orK595A, K595D, K595E relative to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with AsCas12a): K548A, K548D, K548E, N552A, N552D, N552E, orK607A, K607D, K607 relative to the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with CtCas12a): K534A, K534D, K534E, Y538A, Y538D, Y538E, orR591A, R591D, R591E relative to the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with EeCas12a): K542A, K541D, K541E, N545A, N545D, N545E orK601A, K601D, K601E relative to the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with Mb3Cas12a): K579A, K579D, K579E, N583A, N583D, N583E orK635A, K635D, K635E relative to the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with FnCas12a): K613A, K613D, K613E, N617A, N617D, N617E orK671A, K671D, K671E relative to the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with FnoCas12a): K613A, K613D, K613E, N617A, N617D, N617E orN671A, N671D, N671E relative to the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with FbCas12a): K617A, K617D, K617E, N621A, N621D, N621E orK678A, K678D, K678E relative to the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with Lb4Cas12a): K541A, K541D, K541E, N545A, N545D, N545E orK601A, K601D, K601E relative to the amino acid sequence of SEQ ID NO: 9.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with MbCas12a): K569A, K569D, K569E, N573A, N573D, N573E orK625A, K625D, K625E relative to the amino acid sequence of SEQ ID NO:10.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with Pb2Cas12a): K562A, K562D, K562E, N566A, N566D, N566E orK619A, K619D, K619E relative to the amino acid sequence of SEQ ID NO:11.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with PgCas12a): K645A, K645D, K645E, N649A, N649D, N649E orK732A, K732D, K732E relative to the amino acid sequence of SEQ ID NO:12.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with AaCas12a): K548A, K548D, K548E, N552A, N552D, N552E orK607A, K607D, K607E relative to the amino acid sequence of SEQ ID NO:13.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with BoCas12a): K592A, K592D, K592E, N596A, N596D, N596E orK653A, K653D, K653E relative to the amino acid sequence of SEQ ID NO:14.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with CMaCas12a): K521A, K521D, K521E, K525A, K525D, K525E orK577A, K577D, K577E relative to the amino acid sequence of SEQ ID NO:15.

The mutations described herein may be described in the context of anatural Cas12a (any one of SEQ ID NOs: 15) sequence and mutationalpositions can be carried out by aligning the amino acid sequence of aCas12a nucleic acid-guided nuclease with, for example, SEQ ID NO: 1 andmaking the equivalent modification (e.g., substitution) at theequivalent position. By way of example, Table 8 illustrates theequivalent amino acid positions of fifteen orthologous Cas12a nucleicacid-guided nucleases (SEQ ID NOs: 1-15). Any one of the amino acidsindicated in Table 8 may be mutated (i.e., via a comparable amino acidsubstitution).

TABLE 8 Equivalent amino acid positions in homologous Cas12a nucleicacid-guided nuclease Cas 12a AA AA AA AA WT SEQ ID NO Ortholog positionposition position position SEQ ID NO: 1 LbCas12a G532 K538 Y542 K595 SEQID NO: 2 AsCas12a S542 K548 N552 K607 SEQ ID NO: 3 CtCas12a N528 K534Y538 R591 SEQ ID NO: 4 EeCas12a N535 K541 N545 K601 SEQ ID NO: 5Mb3Cas12a N573 K579 N583 K635 SEQ ID NO: 6 FnCas12a N607 K613 N617 K671SEQ ID NO: 7 FnoCas12a N607 K613 N617 N671 SEQ ID NO: 8 FbCas12a N611K617 N621 K678 SEQ ID NO: 9 Lb4Cas12a N535 K541 N545 K601 SEQ ID NO: 10MbCas12a N563 K569 N573 K625 SEQ ID NO: 11 Pb2Cas12a G556 K562 N566 K619SEQ ID NO: 12 PgCas12a D639 K645 N649 K732 SEQ ID NO: 13 AaCas12a S542K548 N552 K607 SEQ ID NO: 14 BoCas12a K586 K592 N596 K653 SEQ ID NO: 15CMaCas12a D515 K521 N525 K577

The variant single-strand-specific Cas12a nucleic acid-guided nucleasesof the disclosure may have at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to any one of SEQ ID NOs:1-15 (excluding the residues listed in Table 8) and contain anyconservative mutation one or more residues indicated in Tables 9-13.

It should be appreciated that any of the amino acid mutations describedherein, (e.g., K595A) from a first amino acid residue (e.g., K, an aminoacid with a basic side chain) to a second amino acid residue (e.g., A,an amino acid with an aliphatic side chain) may also include mutationsfrom the first amino acid residue, lysine, to an amino acid residue thatis similar to (e.g., conserved) the second amino acid residue, alanine,such as valine or glycine. As another example, mutation of an amino acidwith a positively charged side chain (e.g., arginine, histidine, orlysine) may be a mutation to a second amino acid with an acidic sidechain (e.g., glutamic acid or aspartic acid). As another example,mutation of an amino acid with a polar side chain (e.g., serine,threonine, asparagine, or glutamine) may be a mutation to a second aminoacid with a positively charged side chain (e.g., arginine, histidine, orlysine). The skilled artisan would recognize that such conservativeamino acid substitutions will likely have minor effects on proteinstructure and are likely to be well tolerated without compromisingfunction. That is, a mutation from one amino acid to a threonine may bean amino acid mutation to a serine; a mutation from one amino acid to anarginine may be an amino acid mutation to a lysine; a mutation from oneamino acid to an isoleucine, may be an amino acid mutation to analanine, valine, methionine, or leucine; a mutation from one amino acidto a lysine may be an amino acid mutation to an arginine; a mutationfrom one amino acid to an aspartic acid may be an amino acid mutation toa glutamic acid or asparagine; a mutation from one amino acid to avaline may be an amino acid mutation to an alanine, isoleucine,methionine, or leucine; a mutation from one amino acid to a glycine maybe an amino acid mutation to an alanine. It should be appreciated,however, that additional conserved amino acid residues would berecognized by the skilled artisan and any of the amino acid mutations toother conserved amino acid residues are also within the scope of thisdisclosure.

Exemplary variant Cas12a orthologs are shown in tables 9-13.

TABLE 9 Exemplary Variant Ortholog Cas12a's Variant LbCas12a VariantAsCas12a Variant CtCas12a SEQ (in relation to wt SEQ (in relation to wtSEQ (in relation to wt ID LbCas12a SEQ ID ID AsCas12a SEQ ID ID CtCas12aSEQ ID NO: NO: 1) NO: NO: 2) NO: NO: 3) 16 K595A 55 K607A 94 R591A 17K595D 56 K607D 95 R591D 18 K595E 57 K607E 96 R591E 19 K538A/K595A 58K548A/K607A 97 K534A/R591A 20 K538A/K595D 59 K548A/K607D 98 K534A/R591D21 K538A/K595E 60 K548A/K607E 99 K534A/R591E 22 K538D/K595A 61K548D/K607A 100 K534D/R591A 23 K538D/K595D 62 K548D/K607D 101K534D/R591D 24 K538D/K595E 63 K548D/K607E 102 K534D/R591E 25 K538E/K595A64 K548E/K607A 103 K534E/R591A 26 K538E/K595D 65 K548E/K607D 104K534E/R591D 27 K538E/K595E 66 K548E/K607E 105 K534E/R591E 28K538A/Y542A/K595A 67 K548A/N552A/K607A 106 K534A/Y538A/R591A 29K538A/Y542D/K595A 68 K548A/N552D/K607A 107 K534A/Y538D/R591A 30K538A/Y542E/K595A 69 K548A/N552E/K607A 108 K534A/Y538E/R591A 31K538A/Y542A/K595D 70 K548A/N552A/K607D 109 K534A/Y538A/R591D 32K538A/Y542D/K595D 71 K548A/N552D/K607D 110 K534A/Y538D/R591D 33K538A/Y542E/K595D 72 K548A/N552E/K607D 111 K534A/Y538E/R591D 34K538A/Y542A/K595E 73 K548A/N552A/K607E 112 K534A/Y538A/R591E 35K538A/Y542D/K595E 74 K548A/N552D/K607E 113 K534A/Y538D/R591E 36K538A/Y542E/K595E 75 K548A/N552E/K607E 114 K534A/Y538E/R591E 37K538D/Y542A/K595A 76 K548D/N552A/K607A 115 K534D/Y538A/R591A 38K538D/Y542D/K595A 77 K548D/N552D/K607A 116 K534D/Y538D/R591A 39K538D/Y542E/K595A 78 K548D/N552E/K607A 117 K534D/Y538E/R591A 40K538D/Y542A/K595D 79 K548D/N552A/K607D 118 K534D/Y538A/R591D 41K538D/Y542D/K595D 80 K548D/N552D/K607D 119 K534D/Y538D/R591D 42K538D/Y542E/K595D 81 K548D/N552E/K607D 120 K534D/Y538E/R591D 43K538D/Y542A/K595E 82 K548D/N552A/K607E 121 K534D/Y538A/R591E 44K538D/Y542D/K595E 83 K548D/N552D/K607E 122 K534D/Y538D/R591E 45K538D/Y542E/K595E 84 K548D/N552E/K607E 123 K534D/Y538E/R591E 46K538E/Y542A/K595A 85 K548E/N552A/K607A 124 K534E/Y538A/R591A 47K538E/Y542D/K595A 86 K548E/N552D/K607A 125 K534E/Y538D/R591A 48K538E/Y542E/K595A 87 K548E/N552E/K607A 126 K534E/Y538E/R591A 49K538E/Y542A/K595E 88 K548E/N552A/K607D 127 K534E/Y538A/R591D 50K538E/Y542D/K595E 89 K548E/N552D/K607D 128 K534E/Y538D/R591D 51K538E/Y542E/K595E 90 K548E/N552E/K607D 129 K534E/Y538E/R591D 52K538E/Y542A/K595E 91 K548E/N552A/K607E 130 K534E/Y538A/R591E 53K538E/Y542D/K595E 92 K548E/N552D/K607E 131 K534E/Y538D/R591E 54K538E/Y542E/K595E 93 K548E/N552E/K607E 132 K534E/Y538E/R591E

TABLE 10 Exemplary Variant Ortholog Cas12a's Variant EeCas12a VariantMb3Cas12a Variant FnCas12a SEQ (in relation to wt SEQ (in relation to wtSEQ (in relation to wt ID EeCas12a SEQ ID ID Mb3Cas12a SEQ ID IDFnCas12a SEQ ID NO: NO: 4) NO: NO: 5) NO: NO: 6) 133 K601A 172 K635A 211K671A 134 K601D 173 K635D 212 K671D 135 K601E 174 K635E 213 K671E 136K541A/K601A 175 K579A/K635A 214 K613A/K671A 137 K541A/K601D 176K579A/K635D 215 K613A/K671D 138 K541A/K601E 177 K579A/K635E 216K613A/K671E 139 K541D/K601A 178 K579D/K635A 217 K613D/K671A 140K541D/K601D 179 K579D/K635D 218 K613D/K671D 141 K541D/K601E 180K579D/K635E 219 K613D/K671E 142 K541E/K601A 181 K579E/K635A 220K613E/K671A 143 K541E/K601D 182 K579E/K635D 221 K613E/K671D 144K541E/K601E 183 K579E/K635E 222 K613E/K671E 145 K541A/N545A/K601A 184K579A/N583A/K635A 223 K613A/N617A/K671A 146 K541A/N545D/K601A 185K579A/N583D/K635A 224 K613A/N617D/K671A 147 K541A/N545E/K601A 186K579A/N583E/K635A 225 K613A/N617E/K671A 148 K541A/N545A/K601D 187K579A/N583A/K635D 226 K613A/N617A/K671D 149 K541A/N545D/K601D 188K579A/N583D/K635D 227 K613A/N617D/K671D 150 K541A/N545E/K601D 189K579A/N583E/K635D 228 K613A/N617E/K671D 151 K541A/N545A/K601E 190K579A/N583A/K635E 229 K613A/N617A/K671E 152 K541A/N545D/K601E 191K579A/N583D/K635E 230 K613A/N617D/K671E 153 K541A/N545E/K601E 192K579A/N583E/K635E 231 K613A/N617E/K671E 154 K541D/N545A/K601A 193K579D/N583A/K635A 232 K613D/N617A/K671A 155 K541D/N545D/K601A 194K579D/N583D/K635A 233 K613D/N617D/K671A 156 K541D/N545E/K601A 195K579D/N583E/K635A 234 K613D/N617E/K671A 157 K541D/N545A/K601D 196K579D/N583A/K635D 235 K613D/N617A/K671D 158 K541D/N545D/K601D 197K579D/N583D/K635D 236 K613D/N617D/K671D 159 K541D/N545E/K601D 198K579D/N583E/K635D 237 K613D/N617E/K671D 160 K541D/N545A/K601E 199K579D/N583A/K635E 238 K613D/N617A/K671E 161 K541D/N545D/K601E 200K579D/N583D/K635E 239 K613D/N617D/K671E 162 K541D/N545E/K601E 201K579D/N583E/K635E 240 K613D/N617E/K671E 163 K541E/N545A/K601A 202K579E/N583A/K635A 241 K613E/N617A/K671A 164 K541E/N545D/K601A 203K579E/N583D/K635A 242 K613E/N617D/K671A 165 K541E/N545E/K601A 204K579E/N583E/K635A 243 K613E/N617E/K671A 166 K541E/N545A/K601D 205K579E/N583A/K635D 244 K613E/N617A/K671D 167 K541E/N545D/K601D 206K579E/N583D/K635D 245 K613E/N617D/K671D 168 K541E/N545E/K601D 207K579E/N583E/K635D 246 K613E/N617E/K671D 169 K541E/N545A/K601E 208K579E/N583A/K635E 247 K613E/N617A/K671E 170 K541E/N545D/K601E 209K579E/N583D/K635E 248 K613E/N617D/K671E 171 K541E/N545E/K601E 210K579E/N583E/K635E 249 K613E/N617E/K671E

TABLE 11 Exemplary Variant Ortholog Cas12a's Variant FnoCas12a VariantFbCas12a Variant Lb4as12a SEQ (in relation to wt SEQ (in relation to wtSEQ (in relation to wt ID FnoCas12a SEQ ID ID FbCas12a SEQ ID IDLb4Cas12a SEQ ID NO: NO: 7) NO: NO: 8) NO: NO: 9) 250 N671A 289 K678A328 K601A 251 N671D 290 K678D 329 K601D 252 N671E 291 K678E 330 K601E253 K613A/N671A 292 K617A/K678A 331 K541A/K601A 254 K613A/N671D 293K617A/K678D 332 K541A/K601D 255 K613A/N671E 294 K617A/K678E 333K541A/K601E 256 K613D/N671A 295 K617D/K678A 334 K541D/K601A 257K613D/N671D 296 K617D/K678D 335 K541D/K601D 258 K613D/N671E 297K617D/K678E 336 K541D/K601E 259 K613E/N671A 298 K617E/K678A 337K541E/K601A 260 K613E/N671D 299 K617E/K678D 338 K541E/K601D 261K613E/N671E 300 K617E/K678E 339 K541E/K601E 262 K613A/N617A/N671A 301K617A/N621A/K678A 340 K541A/N545A/K601A 263 K613A/N617D/N671A 302K617A/N621D/K678A 341 K541A/N545D/K601A 264 K613A/N617E/N671A 303K617A/N621E/K678A 342 K541A/N545E/K601A 265 K613A/N617A/N671D 304K617A/N621A/K678D 343 K541A/N545A/K601D 266 K613A/N617D/N671D 305K617A/N621D/K678D 344 K541A/N545D/K601D 267 K613A/N617E/N671D 306K617A/N621E/K678D 345 K541A/N545E/K601D 268 K613A/N617A/N671E 307K617A/N621A/K678E 346 K541A/N545A/K601E 269 K613A/N617D/N671E 308K617A/N621D/K678E 347 K541A/N545D/K601E 270 K613A/N617E/N671E 309K617A/N621E/K678E 348 K541A/N545E/K601E 271 K613D/N617A/N671A 310K617D/N621A/K678A 349 K541D/N545A/K601A 272 K613D/N617D/N671A 311K617D/N621D/K678A 350 K541D/N545D/K601A 273 K613D/N617E/N671A 312K617D/N621E/K678A 351 K541D/N545E/K601A 274 K613D/N617A/N671D 313K617D/N621A/K678D 352 K541D/N545A/K601D 275 K613D/N617D/N671D 314K617D/N621D/K678D 353 K541D/N545D/K601D 276 K613D/N617E/N671D 315K617D/N621E/K678D 354 K541D/N545E/K601D 277 K613D/N617A/N671E 316K617D/N621A/K678E 355 K541D/N545A/K601E 278 K613D/N617D/N671E 317K617D/N621D/K678E 356 K541D/N545D/K601E 279 K613D/N617E/N671E 318K617D/N621E/K678E 357 K541D/N545E/K601E 280 K613E/N617A/N671A 319K617E/N621A/K678A 358 K541E/N545A/K601A 281 K613E/N617D/N671A 320K617E/N621D/K678A 359 K541E/N545D/K601A 282 K613E/N617E/N671A 321K617E/N621E/K678A 360 K541E/N545E/K601A 283 K613E/N617A/N671D 322K617E/N621A/K678D 361 K541E/N545A/K601D 284 K613E/N617D/N671D 323K617E/N621D/K678D 362 K541E/N545D/K601D 285 K613E/N617E/N671D 324K617E/N621E/K678D 363 K541E/N545E/K601D 286 K613E/N617A/N671E 325K617E/N621A/K678E 364 K541E/N545A/K601E 287 K613E/N617D/N671E 326K617E/N621D/K678E 365 K541E/N545D/K601E 288 K613E/N617E/N671E 327K617E/N621E/K678E 366 K541E/N545E/K601E

TABLE 12 Exemplary Variant Ortholog Cas12a's Variant MbCas12a VariantPb2Cas12a Variant PgCas12a SEQ (in relation to wt SEQ (in relation to wtSEQ (in relation to wt ID MbCas12a SEQ ID ID Pb2Cas12a SEQ ID IDPgCas12a SEQ ID NO: NO: 10) NO: NO: 11) NO: NO: 12) 367 K625A 406 K619A445 K732A 368 K625D 407 K619D 446 K732D 369 K625E 408 K619E 447 K732E370 K569A/K625A 409 K562A/K619A 448 K645A/K732A 371 K569A/K625D 410K562A/K619D 449 K645A/K732D 372 K569A/K625E 411 K562A/K619E 450K645A/K732E 373 K569D/K625A 412 K562D/K619A 451 K645D/K732A 374K569D/K625D 413 K562D/K619D 452 K645D/K732D 375 K569D/K625E 414K562D/K619E 453 K645D/K732E 376 K569E/K625A 415 K562E/K619A 454K645E/K732A 377 K569E/K625D 416 K562E/K619D 455 K645E/K732D 378K569E/K625E 417 K562E/K619E 456 K645E/K732E 379 K569A/N573A/K625A 418K562A/N566A/K619A 457 K645A/N649A/K732A 380 K569A/N573D/K625A 419K562A/N566D/K619A 458 K645A/N649D/K732A 381 K569A/N573E/K625A 420K562A/N566E/K619A 459 K645A/N649E/K732A 382 K569A/N573A/K625D 421K562A/N566A/K619D 460 K645A/N649A/K732D 383 K569A/N573D/K625D 422K562A/N566D/K619D 461 K645A/N649D/K732D 384 K569A/N573E/K625D 423K562A/N566E/K619D 462 K645A/N649E/K732D 385 K569A/N573A/K625E 424K562A/N566A/K619E 463 K645A/N649A/K732E 386 K569A/N573D/K625E 425K562A/N566D/K619E 464 K645A/N649D/K732E 387 K569A/N573E/K625E 426K562A/N566E/K619E 465 K645A/N649E/K732E 388 K569D/N573A/K625A 427K562D/N566A/K619A 466 K645D/N649A/K732A 389 K569D/N573D/K625A 428K562D/N566D/K619A 467 K645D/N649D/K732A 390 K569D/N573E/K625A 429K562D/N566E/K619A 468 K645D/N649E/K732A 391 K569D/N573A/K625D 430K562D/N566A/K619D 469 K645D/N649A/K732D 392 K569D/N573D/K625D 431K562D/N566D/K619D 470 K645D/N649D/K732D 393 K569D/N573E/K625D 432K562D/N566E/K619D 471 K645D/N649E/K732D 394 K569D/N573A/K625E 433K562D/N566A/K619E 472 K645D/N649A/K732E 395 K569D/N573D/K625E 434K562D/N566D/K619E 473 K645D/N649D/K732E 396 K569D/N573E/K625E 435K562D/N566E/K619E 474 K645D/N649E/K732E 397 K569E/N573A/K625A 436K562E/N566A/K619A 475 K645E/N649A/K732A 398 K569E/N573D/K625A 437K562E/N566D/K619A 476 K645E/N649D/K732A 399 K569E/N573E/K625A 438K562E/N566E/K619A 477 K645E/N649E/K732A 400 K569E/N573A/K625D 439K562E/N566A/K619D 478 K645E/N649A/K732D 401 K569E/N573D/K625D 440K562E/N566D/K619D 479 K645E/N649D/K732D 402 K569E/N573E/K625D 441K562E/N566E/K619D 480 K645E/N649E/K732D 403 K569E/N573A/K625E 442K562E/N566A/K619E 481 K645E/N649A/K732E 404 K569E/N573D/K625E 443K562E/N566D/K619E 482 K645E/N649D/K732E 405 K569E/N573E/K625E 444K562E/N566E/K619E 483 K645E/N649E/K732E

TABLE 13 Exemplary Variant Ortholog Cas12a's Variant AaCas12a VariantBoCas12a Variant CMaCas12a SEQ (in relation to wt SEQ (in relation to wtSEQ (in relation to wt ID AaCas12a SEQ ID ID BoCas12a SEQ ID IDCMaCas12a SEQ ID NO: NO: 13) NO: NO: 14) NO: NO: 15) 484 K607A 523 K653A562 K577A 485 K607D 524 K653D 563 K577D 486 K607E 525 K653E 564 K577E487 K548A/K607A 526 K592A/K653A 565 K521A/K577A 488 K548A/K607D 527K592A/K653D 566 K521A/K577D 489 K548A/K607E 528 K592A/K653E 567K521A/K577E 490 K548D/K607A 529 K592D/K653A 568 K521D/K577A 491K548D/K607D 530 K592D/K653D 569 K521D/K577D 492 K548D/K607E 531K592D/K653E 570 K521D/K577E 493 K548E/K607A 532 K592E/K653A 571K521E/K577A 494 K548E/K607D 533 K592E/K653D 572 K521E/K577D 495K548E/K607E 534 K592E/K653E 573 K521E/K577E 496 K548A/N552A/K607A 535K592A/N596A/K653A 574 K521A/N525A/K577A 497 K548A/N552D/K607A 536K592A/N596D/K653A 575 K521A/N525D/K577A 498 K548A/N552E/K607A 537K592A/N596E/K653A 576 K521A/N525E/K577A 499 K548A/N552A/K607D 538K592A/N596A/K653D 577 K521A/N525A/K577D 500 K548A/N552D/K607D 539K592A/N596D/K653D 578 K521A/N525D/K577D 501 K548A/N552E/K607D 540K592A/N596E/K653D 579 K521A/N525E/K577D 502 K548A/N552A/K607E 541K592A/N596A/K653E 580 K521A/N525A/K577E 503 K548A/N552D/K607E 542K592A/N596D/K653E 581 K521A/N525D/K577E 504 K548A/N552E/K607E 543K592A/N596E/K653E 582 K521A/N525E/K577E 505 K548D/N552A/K607A 544K592D/N596A/K653A 583 K521D/N525A/K577A 506 K548D/N552D/K607A 545K592D/N596D/K653A 584 K521D/N525D/K577A 507 K548D/N552E/K607A 546K592D/N596E/K653A 585 K521D/N525E/K577A 508 K548D/N552A/K607D 547K592D/N596A/K653D 586 K521D/N525A/K577D 509 K548D/N552D/K607D 548K592D/N596D/K653D 587 K521D/N525D/K577D 510 K548D/N552E/K607D 549K592D/N596E/K653D 588 K521D/N525E/K577D 511 K548D/N552A/K607E 550K592D/N596A/K653E 589 K521D/N525A/K577E 512 K548D/N552D/K607E 551K592D/N596D/K653E 590 K521D/N525D/K577E 513 K548D/N552E/K607E 552K592D/N596E/K653E 591 K521D/N525E/K577E 514 K548E/N552A/K607A 553K592E/N596A/K653A 592 K521E/N525A/K577A 515 K548E/N552D/K607A 554K592E/N596D/K653A 593 K521E/N525D/K577A 516 K548E/N552E/K607A 555K592E/N596E/K653A 594 K521E/N525E/K577A 517 K548E/N552A/K607D 556K592E/N596A/K653D 595 K521E/N525A/K577D 518 K548E/N552D/K607D 557K592E/N596D/K653D 596 K521E/N525D/K577D 519 K548E/N552E/K607D 558K592E/N596E/K653D 597 K521E/N525E/K577D 520 K548E/N552A/K607E 559K592E/N596A/K653E 598 K521E/N525A/K577E 521 K548E/N552D/K607E 560K592E/N596D/K653E 599 K521E/N525D/K577E 522 K548E/N552E/K607E 561K592E/N596E/K653E 600 K521E/N525E/K577E

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease is at least 70% identical to any one of SEQ ID NOs:16-600. In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease is at least 75% identical to any one of SEQ ID NOs:16-600 16-600. In some embodiments, the single-strand-specific Cas12anucleic acid-guided nuclease is at least 80% identical to any one of SEQID NOs: 16-600. In some embodiments, the single-strand-specific Cas12anucleic acid-guided nuclease is at least 85% identical to any one of SEQID NOs: 16-600. In some embodiments, the single-strand-specific Cas12anucleic acid-guided nuclease is at least 90% identical to any one of SEQID NOs: 16-600. In some embodiments, the single-strand-specific Cas12anucleic acid-guided nuclease is at least 95% identical to any one of SEQID NOs: 16-600. In some embodiments, the single-strand-specific Cas12anucleic acid-guided nuclease is at least 96%, 97%, 98% or 99% identicalto any one of SEQ ID NOs: 16-600. In some embodiments, thesingle-strand-specific Cas12a nucleic acid-guided nuclease is any one ofSEQ ID NOs: 16-600.

The mutations described herein are described in the context of the WTLbCas12a (e.g., SEQ ID NO: 1) sequence and mutational positions can becarried out by aligning the amino acid sequence of a Cas12a nucleicacid-guided nuclease with SEQ ID NO: 1 and making the equivalentmodification (e.g., substitution) at the equivalent position. By way ofexample, the mutations described herein may be applied to a Cas12aenzyme shown in Table 7, or any other homolog Cas12a thereof by aligningthe amino acid sequence of the Cas12a to SEQ ID NO: 1 and making themodifications described in Tables 9-13 (changes to the wildtype residueto alanine, aspartic acid or glutamic acid or conservative equivalentsat the Cas12a ortholog's equivalent position (e.g., see Table 8 for anexample of equivalent residue positions).

For example, in addition to the variant LbCas12a sequences in Table 9(variant sequences SEQ ID Nos: 16-54), like variants are envisioned forAsCas12a (variant sequences SEQ ID Nos: 55-93), CtCas12a (variantsequences SEQ ID Nos: 94-132), EeCas12a (variant sequences SEQ ID Nos:133-171), Mb3Cas12a (variant sequences SEQ ID Nos: 172-210), FnCas12a(variant sequences SEQ ID Nos: 211-249), FnoCas12a (variant sequencesSEQ ID Nos: 250-288), FbCas12a (variant sequences SEQ ID Nos: 289-327),Lb4Cas12a (variant sequences SEQ ID Nos: 328-366), MbCas12a (variantsequences SEQ ID Nos: 367-405), Pb2Cas12a (variant sequences SEQ ID Nos:406-444), PgCas12a (variant sequences SEQ ID Nos: 445-483), AaCas12a(variant sequences SEQ ID Nos: 484-522), BoCas12a (variant sequences SEQID Nos: 523-561), and CmaCas12a (variant sequences SEQ ID Nos: 562-600).In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease is at least 70% identical to any one of SEQ ID NOs:16-600 and contains an amino acid substitution(s) listed in Tables 9-13or the equivalent in a different ortholog. In some embodiments, thesingle-strand-specific Cas12a nucleic acid-guided nuclease is at least75% identical to any one of SEQ ID NOs: 16-600 and contains an aminoacid substitution(s) listed in Tables 9-13 or the equivalent in adifferent ortholog. In some embodiments, the single-strand-specificCas12a nucleic acid-guided nuclease is at least 80% identical to any oneof SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listedin Tables 9-13 or the equivalent in a different ortholog. In someembodiments, the single-strand-specific Cas12a nucleic acid-guidednuclease is at least 85% identical to any one of SEQ ID NOs: 16-600 andcontains an amino acid substitution(s) listed in Tables 9-13 or theequivalent in a different ortholog. In some embodiments, thesingle-strand-specific Cas12a nucleic acid-guided nuclease is at least90% identical to any one of SEQ ID NOs: 16-600 and contains an aminoacid substitution(s) listed in Tables 9-13 or the equivalent in adifferent ortholog. In some embodiments, the single-strand-specificCas12a nucleic acid-guided nuclease is at least 95% identical to any oneof SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listedin Tables 9-13 or the equivalent in a different ortholog. In someembodiments, the single-strand-specific Cas12a nucleic acid-guidednuclease is at least %, 97%, 98% or 99% identical to any one of SEQ IDNOs: 16-600 and contains an amino acid substitution(s) listed in Tables9-13 or the equivalent in a different ortholog. In some embodiments, thesingle-strand-specific Cas12a nucleic acid-guided nuclease is any one ofSEQ ID NOs: 16-600.

The single-strand-specific Cas12a nucleic acid-guided nucleasesdescribed herein may be any Cas12a nucleic acid-guided nuclease thatlargely prevents double-stranded nucleic acid unwinding and R-loopformation. The single-strand-specific Cas12a nucleic acid-guidednucleases described herein may also be any Cas12a nucleic acid-guidednuclease that lacks cis-cleavage activity yet maintains trans-nucleicacid-guided nuclease activity on single-stranded nucleic acid molecules.Such single-strand-specific Cas12a nucleic acid-guided nucleases may begenerated via the mutations described herein.

Additionally, or alternatively, such single-strand-specific Cas12anucleic acid-guided nucleases may be generated via post-translationalmodifications (e.g., acetylation). The single-strand-specific Cas12anucleic acid-guided nucleases of the disclosure may be an acetylatedCas12a enzyme. The single-strand-specific Cas12a nucleic acid-guidednucleases of the disclosure may be an LbCas12a (i.e., SEQ ID NO: 1) withan acetylated K595 (K595K^(Ac)) residue. The single-strand-specificCas12a nucleic acid-guided nucleases of the disclosure may be anAsCas12a (i.e., SEQ ID NO: 2) with an acetylated K607 (K607K^(Ac))residue. The single-strand-specific Cas12a nucleic acid-guided nucleasesof the disclosure may be a CtCas12a (i.e., SEQ ID NO: 3) with anacetylated R591 (R591R A c) residue. The single-strand-specific Cas12anucleic acid-guided nucleases of the disclosure may be an EeCas12a(i.e., SEQ ID NO: 4) with an acetylated K601 (K607K^(Ac)) residue. Thesingle-strand-specific Cas12a nucleic acid-guided nucleases of thedisclosure may be an Mb3Cas12a (i.e., SEQ ID NO: 5) with an acetylatedK635 (K635K^(Ac)) residue. The single-strand-specific Cas12a nucleicacid-guided nucleases of the disclosure may be an FnCas12a (i.e., SEQ IDNO: 6) with an acetylated K671 (K671K^(Ac)) residue. Thesingle-strand-specific Cas12a nucleic acid-guided nucleases of thedisclosure may be an FnoCas12a (i.e., SEQ ID NO: 7) with an acetylatedN671 (N671K^(Ac)) residue. The single-strand-specific Cas12a nucleicacid-guided nucleases of the disclosure may be an FbCas12a (i.e., SEQ IDNO: 8) with an acetylated K678 (K678K^(Ac)) residue. Thesingle-strand-specific Cas12a nucleic acid-guided nucleases of thedisclosure may be an Lb4Cas12a (i.e., SEQ ID NO: 9) with an acetylatedK601 (K601K^(Ac)) residue. The single-strand-specific Cas12a nucleicacid-guided nucleases of the disclosure may be an MbCas12a (i.e., SEQ IDNO: 10) with an acetylated K625 (K625K^(Ac)) residue. Thesingle-strand-specific Cas12a nucleic acid-guided nucleases of thedisclosure may be a Pb2Cas12a (i.e., SEQ ID NO: 11) with an acetylatedK619 (K619K^(Ac)) residue. The single-strand-specific Cas12a nucleicacid-guided nucleases of the disclosure may be a PgCas12a (i.e., SEQ IDNO: 12) with an acetylated K732 (K732K^(Ac)) residue. Thesingle-strand-specific Cas12a nucleic acid-guided nucleases of thedisclosure may be an AaCas12a (i.e., SEQ ID NO: 13) with an acetylatedK607 (K607K^(Ac)) residue. The single-strand-specific Cas12a nucleicacid-guided nucleases of the disclosure may be an BoCas12a (i.e., SEQ IDNO: 14) with an acetylated K653 (K653K^(Ac)) residue. Thesingle-strand-specific Cas12a nucleic acid-guided nucleases of thedisclosure may be an CmaCas12a (i.e., SEQ ID NO: 15) with an acetylatedK577 (K577K^(Ac)) residue. The single-strand-specific Cas12a nucleicacid-guided nucleases of the disclosure may be a Cas12a orthologacetylated at the amino acid of the ortholog equivalent to K595 of SEQID NO:1. Acetylation of Cas12a can be carried out with any suitableacetyltransferase. For a discussion and methods for disabling of Cas12aby ArVA5, see Dong, et al., Nature Structural and Molecular Bio.,26(4):308-14 (2019). For example, LbCas12a can be incubated with AcrVA5in order to acetylate the K595 residue, thereby deactivating the dsDNAactivity (e.g., FIG. 7 ). In addition to acetylation, phosphorylationand methylation of select amino acid residues may be employed.

Bulky Modifications

In addition to the modalities of adjusting the ratio of theconcentration of the blocked nucleic acid molecules to the concentrationof the RNP2 and altering the domains of the variant nucleic acid-guidednuclease of RNP2 that interact with the PAM region or surroundingsequences on the blocked nucleic acid molecules to vary dsDNA vs. ssDNArecognition properties as described in detail above, the presentdisclosure additionally contemplates use of “bulky modifications” at the5′ and/or 3′ ends and/or at internal nucleic acid bases of the blockednucleic acid molecule and/or using modifications between internalnucleic acid bases. FIG. 8A is an illustration of the steric hindranceat the PAM-interacting (PI) domain in a nucleic acid-guided nucleasecaused by 5′ and 3′ modifications to a blocked nucleic acid molecule. Attop in FIG. 8A is an illustration of the target stand and non-targetstrand, and below this is an illustration of a self-hybridized blockednucleic acid molecule comprising three loop regions, as well as bulkymodifications on the 5′ and 3′ ends of the blocked nucleic acidmolecule. Example “bulky modifications” include a fluorophore andquencher pair (as shown here) or biotin, but in general encompassmolecules with a size of about 1 nm or less, or 0.9 nm or less, or 0.8nm or less, or 0.7 nm or less, or 0.6 nm or less, or 0.5 nm or less, or0.4 nm or less, or 0.3 nm or less, or 0.2 nm or less, or 0.1 nm or less,or 0.05 nm or less, or as small as 0.025 nm or less.

In the illustration at center, the blocked nucleic acid molecule withthe 5′ and 3′ ends comprising a fluorophore and a quencher is shownbeing cleaved at the loop regions. Note that the bulky modifications inthis embodiment also allow the blocked nucleic acid molecule to act as areporter moiety; that is, when the loop regions of the blocked nucleicacid molecule are cleaved, the short nucleotide segments of thenon-target strand dehybridize from the target strand due to low T_(m),thereby separating the fluorophore and quencher such that fluorescencefrom the fluorophore is no longer quenched and can be detected. In theillustration at bottom, the intact blocked nucleic acid molecule withthe bulky modifications (at left) sterically hinders interaction withthe PAM-interacting (PI) domain of the nucleic acid-guided nuclease inRNP2 such that the intact blocked nucleic acid molecule cannot becleaved via cis-cleavage by the nucleic acid-guided nuclease. However,once the loop regions of the blocked nucleic acid molecule are cleaved(via, e.g., trans-cleavage from RNP1 (at right)) and the shortnucleotide segments of the non-target strand dehybridize from the targetstrand, leaving the 3′ end of the now single-stranded target strand isnow free to initiate R-loop formation with RNP2. R-loop formation leadsto cis-cleavage of the single-strand target strand, and subsequentactivation of trans-cleavage of RNP2.

FIG. 8B illustrates five exemplary variations of blocked nucleic acidmolecules with bulky modifications, including at the 5′ and/or 3′ endsof a self-hybridizing blocked nucleic acid molecule and/or at internalnucleic acid bases of the blocked nucleic acid molecule. Embodiment (i)illustrates a self-hybridizing blocked nucleic acid molecule having afluorophore at its 5′ end and a quencher at its 3′ end. Embodiment (ii)illustrates a self-hybridizing blocked nucleic acid molecule having afluorophore and a quencher at internal nucleic acid bases flanking aloop sequence. Embodiment (iii) illustrates a self-hybridizing blockednucleic acid molecule having a fluorophore at its 5′ end and a quencherat its 3′ end as well as having a fluorophore and a quencher at internalnucleic acid bases where the internal fluorophore and quencher flank aloop sequence. The fluorophore/quencher embodiments work as long as thefluorophore and quencher are at a distance of about 10-11 nm or lessapart. Embodiment (iv) illustrates a self-hybridizing blocked nucleicacid molecule having a biotin molecule at its 5′ end, and embodiment (v)illustrates a self-hybridizing blocked nucleic acid molecule having abiotin at an internal nucleic acid base. Note that bulky modificationsof internal nucleic acid bases often are made at or near a loop regionof a blocked nucleic acid molecule (or blocked target molecule). Theloop regions are regions of the blocked nucleic acid molecules—inaddition to the 5′ and 3′ ends—that may be vulnerable to unwinding.

Modifications can be used in self-hybridized blocked nucleic acidmolecules lacking a PAM or those comprising a PAM, partiallyself-hybridized blocked nucleic acid molecules lacking a PAM or thosecomprising a PAM, or reverse PAM molecules. Other variations includeusing RNA loops instead of DNA loops if a Cas 13 nucleic acid-guidednuclease is used as the nucleic acid-guided nuclease in RNP1, or entireRNA molecules if a Cas 13 nucleic acid-guided nuclease is used as thenucleic acid-guided nuclease in RNP1 and RNP2.

FIGS. 8C, 8D and 8E list exemplary bulky modifications for 5′, 3′, andinternal positions in blocked nucleic acid molecules, and Table 14 belowlists sequences of exemplary self-hybridizing blocked nucleic acidmolecules. 56-FAM stands for 5′ 6-FAM (6-fluorescein amidite); and 3BHQstands for 3′ BLACK HOLE QUENCHER®-1.

TABLE 14 Bulky Modifications SEQ ID Molecule No. NO: NameMolecule Sequence (5′→3′) 5′ FAM + 3′ BHQ  1 601 5′F_U29_Q /56-FAM/GATCCATTTTATTTTAGAT CATATATATACATGATCGGATC/ 3BHQ_1/  2 602 5′F_1C/56- armor_U29 FAM/CGATCCATTTTATTTTAGAT _Q CATATATATACATGATCGGATCG/3BHQ_1/  3 603 5′F_2CC /56- armor_U29 FAM/CCGATCCATTTTATTTTAGAT QCATATATATACATGATCGGATCGG/ 3BHQ_1/  4 604 5′F_1A /56- armor_U29FAM/AGATCCATTTTATTTTAGAT Q CATATATATACATGATCGGATCT/ 3BHQ_1/  5 6055′F_2AT /56- armor_U29 FAM/ATGATCCATTTTATTTTAGAT _QCATATATATACATGATCGGATCAT/ 3BHQ_1/  6 606 5′F_U250_ /56- QFAM/GATATATAAAAAAAAAAAGAT CATATACATATATGATCATATATC/ 3BHQ_1/  7 6075′F_1C /56- armor_U25 FAM/CGATATATAAAAAAAAAAAGAT 0_QCATATACATATATGATCATATATCG/ 3BHQ_1/  8 608 5′F_2CC /56- armor_U25FAM/CCGATATATAAAAAAAAAAAGAT 0_Q CATATACATATATGATCATATATCGG/ 3BHQ_1/  9609 5′F_1A /56- armor_U25 FAM/AGATATATAAAAAAAAAAAGAT 0_QCATATACATATATGATCATATATCT/ 3BHQ_1/ 10 610 5′F_2AT /56- armor_U25FAM/ATGATATATAAAAAAAAAAAGAT 0_Q CATATACATATATGATCATATATCAT/ 3BHQ_1/5′ Fluorsceine (modification on base) + 3′ BHQ 11 611 5′FdT_U29/5FluorT/GATCCATTTTATTTTAGAT _Q CATATATATACATGATCGGATCA/ 3BHQ_1/ 12 6125′FdT_1C /5FluorT/CGATCCATTTTATTTTAGAT armor_U29CATATATATACATGATCGGATCGA/ _Q 3BHQ_1/ 13 605 5′FdT_1AA/iFluorT/GATCCATTTTATTTTAGAT armor_U29 CATATATATACATGATCGGATCAT/ _Q3BHQ_1/ 14 613 5′FdT_U25 /5FluorT/GATATATAAAAAAAAAAAGAT 0_QCATATACATATATGATCATATATCA/ 3BHQ_1/ 15 614 5′FdT_1C/5FluorT/CGATATATAAAAAAAAAAAGAT armor_U25 CATATACATATATGATCATATATCGA/0_Q 3BHQ_1/ 16 610 5′FdT_1A A/iFluorT/GATATATAAAAAAAAAAAGAT armor_U25CATATACATATATGATCATATATCAT/ 0_Q 3BHQ_1/ 5′ FAM + Internal Fluorsceine (modification on base) + 3′ BHQ 17 601 5′F_IntFdt_ /56- U29_QFAM/GA/iFluorT/CCATTTTATTTTAGAT CATATATATACATGATCGGATC/ 3BHQ_1/ 18 6065′F_IntFdt_ /56- U250_Q FAM/GA/iFluorT/ATATAAAAAAAAAAAGATCATATACATATATGATCATATATC/ 3BHQ_1/ 19 602 5′F_1C /56- armor_IntFFAM/CGA/iFluorT/CCATTTTATTTTA dt_U29_Q GATCATATATATACATGATCGGATCG/3BHQ_1/ 20 604 5′F_1A /56- armor_IntF FAM/AGA/iFluorT/CCATTTTATTTTAdt_U29_Q GATCATATATATACATGATCGGATCT/ 3BHQ_1/ 21 607 5′F_1C /56-armor_IntF FAM/CGA/iFluorT/ATATAAAAAAAAAAA dt_U250_QGATCATATACATATATGATCATATATCG/ 3BHQ_1/ 22 609 5′F_1A /56- armor_IntFFAM/AGA/iFluorT/ATATAAAAAAAAAAA dt_U250_Q GATCATATACATATATGATCATATATCT/3BHQ_1/ 23 603 5′F_2CC /56- armor_IntF FAM/CCGA/iFluorT/CCATTTTATTTTAdt_U29_Q GATCATATATATACATGATCGGATCGG/ 3BHQ_1/ 24 605 5′F_2AT /56-armor_IntF FAM/ATGA/iFluorT/CCATTTTATTTTA dt_U29_QGATCATATATATACATGATCGGATCAT/ 3BHQ_1/ 25 608 5′F_2CC /56- armor_IntFFAM/CCGA/iFluorT/ATATAAAAAAAAAA dt_U250_QAGATCATATACATATATGATCATATATCGG/ 3BHQ_1/ 26 610 5′F_2AT /56- armor_IntFFAM/ATGA/iFluorT/ATATAAAAAAAAAA dt_U250_QAGATCATATACATATATGATCATATATCAT/ 3BHQ_1/

Applications of the Cascade Assay

The present disclosure describes cascade assays for detecting a targetnucleic acid of interest in a sample that provide instantaneous ornearly instantaneous results even at ambient temperatures at 16° C. andabove, allow for massive multiplexing and minimum workflow, yet provideaccurate results at low cost. Moreover, the various embodiments of thecascade assay are notable in that, with the exception of the gRNA inRNP1, the cascade assay components stay the same no matter what targetnucleic acid(s) of interest are being detected and RNP1 is easilyreprogrammed. Moreover, the cascade assay can be massively multiplexedfor detecting several to many to target nucleic acid moleculessimultaneously. For example, the assay may be designed to detect one toseveral to many different pathogens (e.g., testing for many differentpathogens in one assay), or the assay may be designed to detect one toseveral to many different sequences from the same pathogen (e.g., toincrease specificity and sensitivity), or a combination of the two.

As described above, early and accurate identification of, e.g.,infectious agents, microbe contamination, and variant nucleic acidsequences that indicate the present of such diseases such as cancer orcontamination by heterologous sources is important in order to selectcorrect therapeutic treatment, identify tainted food, pharmaceuticals,cosmetics and other commercial goods; and to monitor the environment.The cascade assay described herein can be applied in diagnostics for,e.g., infectious disease (including but not limited to Covid, HIV, flu,the common cold, Lyme disease, STDs, chicken pox, diptheria,mononucleosis, hepatitis, UTIs, pneumonia, tetanus, rabies, malaria,dengue fever, Ebola, plague; see Table 1), for rapid liquid biopsies andcompanion diagnostics (biomarkers for cancers, early detection,progression, monitoring; see Table 4), prenatal testing (including butnot limited to chromosomal abnormalities and genetic diseases such assickle cell, including over-the-counter versions of prenatal testingassays), rare disease testing (achondroplasia, Addison's disease,al-antitrypsin deficiency, multiple sclerosis, muscular dystrophy,cystic fibrosis, blood factor deficiencies), SNP detection/DNAprofiling/epigenetics, genotyping, low abundance transcript detection,labeling for cell or droplet sorting, in situ nucleic acid detection,sample prep, library quantification of NGS, screening biologics(including engineered therapeutic cells for genetic integrity and/orcontamination), development of agricultural products, food compliancetesting and quality control (e.g., detection of genetically modifiedproducts, confirmation of source for high value commodities,contamination detection), infectious disease in livestock, infectiousdisease in cash crops, livestock breeding, drug screening, personalgenome testing including clinical trial stratification, personalizedmedicine, nutrigenomics, drug development and drug therapy efficacy,transplant compatibility and monitoring, environmental testing andforensics, and bioterrorism agent monitoring.

Target nucleic acids of interest are derived from samples as describedin more detail above. Suitable samples for testing include, but are notlimited to, any environmental sample, such as air, water, soil, surface,food, clinical sites and products, industrial sites and products,pharmaceuticals, medical devices, nutraceuticals, cosmetics, personalcare products, agricultural equipment and sites, and commercial samples,and any biological sample obtained from an organism or a part thereof,such as a plant, animal, or microbe. In some embodiments, the biologicalsample is obtained from an animal subject, such as a human subject. Abiological sample may be any solid or fluid sample obtained from,excreted by or secreted by any living organism, including, withoutlimitation, single celled organisms, such as bacteria, yeast,protozoans, and amoebas among others, multicellular organisms includingplants or animals, including samples from a healthy or apparentlyhealthy human subject or a human patient affected by a condition ordisease to be diagnosed or investigated, such as an infection with apathogenic microorganism, such as a pathogenic bacteria or virus.

For example, a biological sample can be a biological fluid obtained froma human or non-human (e.g., livestock, pets, wildlife) animal, and mayinclude but is not limited to blood, plasma, serum, urine, stool,sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleuraleffusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreoushumor, or any bodily secretion, a transudate, an exudate (for example,fluid obtained from an abscess or any other site of infection orinflammation), or fluid obtained from a joint (for example, a normaljoint or a joint affected by disease, such as rheumatoid arthritis,osteoarthritis, gout or septic arthritis), or a swab of skin or mucosalmembrane surface (e.g., a nasal or buccal swab).

In some embodiments, the sample can be a viral or bacterial sample or abiological sample that has been minimally processed, e.g., only treatedwith a brief lysis step prior to detection. In other embodiments,minimal processing can include thermal lysis at an elevated temperatureto release nucleic acids. Suitable methods are contemplated in U.S. Pat.No. 9,493,736, among other references. Common methods for cell lysisinvolve thermal, chemical, enzymatic, or mechanical treatment of thesample or a combination of those (see, e.g., Example I below). In someembodiments, minimal processing can include treating the sample withchaotropic salts such as guanidine isothiocyanate or guanidine HCl.Suitable methods are contemplated in U.S. Pat. Nos. 8,809,519 and7,893,251, among other references. In some embodiments, minimalprocessing may include contacting the sample with reducing agents suchas DTT or TCEP and EDTA to inactivate inhibitors and/or other nucleasespresent in the crude samples. In other embodiments, minimal processingfor biofluids may include centrifuging the samples to obtain cell-debrisfree supernatant before applying the reagents. Suitable methods arecontemplated in U.S. Pat. No. 8,809,519, among other references. Instill other embodiments, minimal processing may include performingDNA/RNA extraction to get purified nucleic acids before applying CRISPRCascade reagents.

Table 15 below lists exemplary commercial sample processing kits, andTable 16 below lists point of care processing techniques.

TABLE 15 Exemplary Commercial Sample and Nucleic Acid Processing KitsManufacturer Kit Sample Type Output Lysing and extraction methodsQiagen ® DNeasy ™ Blood small volumes genomic Isolation of Genomic DNAfrom Small & Tissue Kits of blood DNA Volumes of Blood dried blood 1.Uses Chemical and spots Biological/Enzymatic lysis methods urine 2. UsesSPE with Column Purification tissues Isolation of Genomic DNA fromTissues laser- 1. Uses Chemical and microdissected Biological/Enzymaticlysis methods tissues 2. Used to dissolve and lyse tissue sectionscompletely, higher temperature and longer time incubations up to 24hours are used Qiagen ® QIAamp ® UCP whole blood microbial Specificpretreatment protocols are Pathogen swabs DNA suggested depending onsample type with Mini Handbook cultures -- or without the use of kitsfor Mechanical microbial DNA pelleted Lysis Method before downstreampurification microbial cells applications. body fluids Downstreamapplications contain: 1. Chemical and Biological/Enzymatic lysis methods2. SPE with Column Purification Qiagen ® QIAamp ® Viral plasma and viralDNA 1. Uses Chemical lysis methods RNA Kits serum 2. Uses SPE withColumn Purification CSF urine other cell-free body fluids cell-culturesupernatants swabs Zymo Quick- whole blood genomic 1. Uses chemicallysis methods Research TM DNATMMicroprep plasma DNA 2. Uses SPE withcolumn purification Kit serum body fluids buffy coat lymphocytes swabscultured cells Zymo Quick-DNATM A. fumigatus Microbial Uses Bead lysisand pretreatment with: Research TM Fungal/Bacterial C. albicans DNAMiniprep Kit N. crassa S. cerevisiae 1. Chemical lysis methods with S.pombe chaotropic salts mycelium 2. NAE with SPE with silica matricesGram positive bacteria Gram negative bacteria

TABLE 16 Point of Care Sample Processing Techniques Steps ProtocolExample 1 Protocol Example 2 Protocol Example 3 Field-deployable viralStreamlined Lucira Health  ™M diagnostics using inactivation,CRISPR-Cas13 amplification, and Science, Cas13-based detection ofSARS-COV-2 27; 360(6387): 444-448 Nat Commun, 11: 5921 (2018) (2020) 1.Cell disruption Samples were thermally A NP swab or saliva Lucira Healthuses a (lysis) and treated at ~40° C. for ~15 sample was lysed andsingle buffer that lyses inactivation of minutes for nucleaseinactivated for 10 and inactivates nucleases deactivation, thereafterminutes with thermal nucleases and/or In POC setting, cell at 90° C. for5 minutes treatment. These inhibitors. disruption and for viraldeactivation. samples were incubated A nasal swab is directlyinactivation of Sample Types: for 5 min at 40° C., added to a singlenucleases is done Urine followed by 5 min at lysing/reaction buffercommonly through Saliva 70° C. (or 5 min at 95° C., and vigorouslystirred thermal lysis. Diluted blood if saliva) to release the viral(1:3 with PBS) particulates from the Targets: Viruses swab. Target:SARS-Cov-2 2. Assay on crude Thermally treated Thermally treatedProcessed biological sample biological biological sample is used in anThis is usually a direct samples(above) were samples(above) wereisothermal reaction for assay on the crude used directly for useddirectly for pathogenic nucleic acid sample post cell amplification andamplification and detection. disruption and detection of pathogenicdetection of pathogenic inactivation of nucleic acid. nucleic acid.nucleases. No extraction is usually performed.

FIG. 9 shows a lateral flow assay (LFA) device that can be used todetect the cleavage and separation of a signal from a reporter moiety.For example, the reporter moiety may be a single-stranded ordouble-stranded oligonucleotide with terminal biotin and fluoresceinamidite (FAM) modifications; and, as described above, the reportermoiety may also be part of a blocked nucleic acid. The LFA device mayinclude a pad with binding particles, such as gold nanoparticlesfunctionalized with anti-FAM antibodies; a control line with a firstbinding moiety attached, such as avidin or streptavidin; a test linewith a second binding moiety attached, such as antibodies; and anabsorption pad. After completion of a cascade assay (see FIGS. 2A, 3A,and 3B), the assay reaction mix is added to the pad containing thebinding particles, (e.g., antibody labeled gold nanoparticles). When thetarget nucleic acid of interest is present, a reporter moiety iscleaved, and when the target nucleic acid of interest is absent, thereporter is not cleaved.

A moiety on the reporter binds to the binding particles and istransported to the control line. When the target nucleic acid ofinterest is absent, the reporter moiety is not cleaved, and the firstbinding moiety binds to the reporter moiety, with the binding particlesattached. When the target nucleic acid of interest is present, oneportion of the cleaved reporter moiety binds to the first bindingmoiety, and another portion of the cleaved reporter moiety bound to thebinding particles via the moiety binds to the second binding moiety. Inone example, anti-FAM gold nanoparticles bind to a FAM terminus of areporter moiety and flow sequentially toward the control line and thento the test line. For reporters that are not trans-cleaved, goldnanoparticles attach to the control line via biotin-streptavidin andresult in a dark control line. In a negative test, since the reporterhas not been cleaved, all gold conjugates are trapped on control linedue to attachment via biotin-streptavidin. A negative test will resultin a dark control line with a blank test line. In a positive test,reporter moieties have been trans-cleaved by the cascade assay, therebyseparating the biotin terminus from the FAM terminus. For cleavedreporter moieties, nanoparticles are captured at the test line due toanti-FAM antibodies. This positive test results in a dark test line inaddition to a dark control line.

The components of the cascade assay may be provided in various kits fortesting at, e.g., point of care facilities, in the field, pandemictesting sites, and the like. In one aspect, the kit for detecting atarget nucleic acid of interest in a sample includes: firstribonucleoprotein complexes (RNP1s), second ribonucleoprotein complexes(RNP2s), blocked nucleic acid molecules, and reporter moieties. Thefirst complex (RNP1) comprises a first nucleic acid-guided nuclease anda first gRNA, where the first gRNA includes a sequence complementary tothe target nucleic acid(s) of interest. Binding of the first complex(RNP1) to the target nucleic acid(s) of interest activatestrans-cleavage activity of the first nucleic acid-guided nuclease. Thesecond complex (RNP2) comprises a second nucleic acid-guided nucleaseand a second gRNA that is not complementary to the target nucleic acidof interest. The blocked nucleic acid molecule comprises a sequencecomplementary to the second gRNA, where trans-cleavage of the blockednucleic acid molecule results in an unblocked nucleic acid molecule andthe unblocked nucleic acid molecule can bind to the second complex(RNP2), thereby activating the trans-cleavage activity of the secondnucleic acid-guided nuclease. Activating trans-cleavage activity in RNP2results in an exponential increase in unblocked nucleic acid moleculesand in active reporter moieties, where reporter moieties are nucleicacid molecules and/or are operably linked to the blocked nucleic acidmolecules and produce a detectable signal upon cleavage by RNP2.

In a second aspect, the kit for detecting a target nucleic acid moleculein sample includes: first ribonucleoprotein complexes (RNP1s), secondribonucleoprotein complexes (RNP2s), template molecules, blocked primermolecules, a polymerase, NTPs, and reporter moieties. The firstribonucleoprotein complex (RNP1) comprises a first nucleic acid-guidednuclease and a first gRNA, where the first gRNA includes a sequencecomplementary to the target nucleic acid of interest and where bindingof RNP1 to the target nucleic acid(s) of interest activatestrans-cleavage activity of the first nucleic acid-guided nuclease. Thesecond complex (RNP2) comprises a second nucleic acid-guided nucleaseand a second gRNA that is not complementary to the target nucleic acidof interest. The template molecules comprise a primer binding domain(PBD) sequence as well as a sequence corresponding to a spacer sequenceof the second gRNA. The blocked primer molecules comprise a sequencethat is complementary to the PBD on the template nucleic acid moleculeand a blocking moiety.

Upon binding to the target nucleic acid of interest, RNP1 becomes activetriggering trans-cleavage activity that cuts at least one of the blockedprimer molecules to produce at least one unblocked primer molecule. Theunblocked primer molecule hybridizes to the PBD of one of the templatenucleic acid molecules, is trimmed of excess nucleotides by the 3′-to-5′exonuclease activity of the polymerase and is then extended by thepolymerase and NTPs to form a synthesized activating molecule with asequence that is complementary to the second gRNA of RNP2 (i.e., thesynthesized activating molecule is the target strand). Upon activatingRNP2, additional trans-cleavage activity is initiated, cleaving at leastone additional blocked primer molecule. Continued cleavage of blockedprimer molecules and subsequent activation of more RNP2s proceeds at anexponential rate. A signal is generated upon cleavage of a reportermolecule by active RNP2 complexes; therefore, a change in signalproduction indicates the presence of the target nucleic acid molecule.

Any of the kits described herein may further include a sample collectiondevice, e.g., a syringe, lancet, nasal swab, or buccal swab forcollecting a biological sample from a subject, and/or a samplepreparation reagent, e.g., a lysis reagent. Each component of the kitmay be in separate container or two or more components may be in thesame container. The kit may further include a lateral flow device usedfor contacting the biological sample with the reaction mixture, where asignal is generated to indicate the presence or absence of the targetnucleic acid molecule of interest. In addition, the kit may furtherinclude instructions for use and other information.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example I: Preparation of Nucleic Acids of Interest

Mechanical lysis: Nucleic acids of interest may be isolated by variousmethods depending on the cell type and source (e.g., tissue, blood,saliva, environmental sample, etc.). Mechanical lysis is a widely usedcell lysis method and may be used to extract nucleic acids frombacterial, yeast, plant and mammalian cells. Cells are disrupted byagitating a cell suspension with “beads” at high speeds (beads fordisrupting various types of cells can be sourced from, e.g., OPSDiagnostics (Lebanon NJ, US) and MP Biomedicals (Irvine, CA, USA)).Mechanical lysis via beads begins with harvesting cells in a tissue orliquid, where the cells are first centrifuged and pelleted. Thesupernatant is removed and replaced with a buffer containing detergentsas well as lysozyme and protease. The cell suspension is mixed topromote breakdown of the proteins in the cells and the cell suspensionthen is combined with small beads (e.g., glass, steel, or ceramic beads)that are mixed (e.g., vortexed) with the cell suspension at high speeds.The beads collide with the cells, breaking open the cell membrane withshear forces. After “bead beating”, the cell suspension is centrifugedto pellet the cellular debris and beads, and the supernatant may bepurified via a nucleic acid binding column (such as the MagMAX™Viral/Pathogen Nucleic Acid Isolation Kit from ThermoFisher (Waltham,MA, USA) and others from Qiagen (Hilden, Germany), TakaraBio (San Jose,CA, USA), and Biocomma (Shenzen, China)) to collect the nucleic acids(see the discussion of solid phase extraction below).

Solid phase extraction (SPE): Another method for capturing nucleic acidsis through solid phase extraction. SPE involves a liquid and stationaryphase, which selectively separates the target analyte (here, nucleicacids) from the liquid in which the cells are suspended based onspecific hydrophobic, polar, and/or ionic properties of the targetanalyte in the liquid and the stationary solid matrix. Silica bindingcolumns and their derivatives are the most commonly used SPE techniques,having a high binding affinity for DNA under alkaline conditions andincreased salt concentration; thus, a highly alkaline and concentratedsalt buffer is used. The nucleic acid sample is centrifuged through acolumn with a highly porous and high surface area silica matrix, wherebinding occurs via the affinity between negatively charged nucleic acidsand positively charged silica material. The nucleic acids bind to thesilica matrices, while the other cell components and chemicals passthrough the matrix without binding. One or more wash steps typically areperformed after the initial sample binding (i.e., the nucleic acids tothe matrix), to further purify the bound nucleic acids, removing excesschemicals and cellular components non-specifically bound to the silicamatrix. Alternative versions of SPE include reverse SPE and ion exchangeSPE, and use of glass particles, cellulose matrices, and magnetic beads.

Thermal lysis: Thermal lysis involves heating a sample of mammaliancells, virions, or bacterial cells at high temperatures thereby damagingthe cellular membranes by denaturizing the membrane proteins.Denaturizing the membrane proteins results in the release ofintracellular DNA. Cells are generally heated above 90° C., however timeand temperature may vary depending on sample volume and sample type.Once lysed, typically one or more downstream methods, such as use ofnucleic acid binding columns for solid phase extraction as describedabove, are required to further purify the nucleic acids.

Physical lysis: Common physical lysis methods include sonication andosmotic shock. Sonication involves creating and rupturing of cavities orbubbles to release shockwaves, thereby disintegrating the cellularmembranes of the cells. In the sonication process, cells are added intolysis buffer, often containing phenylmethylsulfonyl fluoride, to inhibitproteases. The cell samples are then placed in a water bath and asonication wand is placed directly into the sample solution. Sonicationtypically occurs between 20-50 kHz, causing cavities to be formedthroughout the solution as a result of the ultrasonic vibrations;subsequent reduction of pressure then causes the collapse of the cavityor bubble resulting in a large amount of mechanical energy beingreleased in the form of a shockwave that propagates through the solutionand disintegrates the cellular membrane. The duration of the sonicationpulses and number of pulses performed varies depending on cell type andthe downstream application. After sonication, the cell suspensiontypically is centrifuged to pellet the cellular debris and thesupernatant containing the nucleic acids may be further purified bysolid phase extraction as described above.

Another form of physical lysis is osmotic shock, which is most typicallyused with mammalian cells. Osmotic shock involves placing cells inDI/distilled water with no salt added. Because the salt concentration islower in the solution than in the cells, water is forced into the cellcausing the cell to burst, thereby rupturing the cellular membrane. Thesample is typically purified and extracted by techniques such as e.g.,solid phase extraction or other techniques known to those of skill inthe art.

Chemical lysis: Chemical lysis involves rupturing cellular and nuclearmembranes by disrupting the hydrophobic-hydrophilic interactions in themembrane bilayers via detergents. Salts and buffers (such as, e.g.,Tris-HCl pH8) are used to stabilize pH during extraction, and chelatingagents (such as ethylenediaminetetraacetic acid (EDTA)) and inhibitors(e.g., Proteinase K) are also added to preserve the integrity of thenucleic acids and protect against degradation. Often, chemical lysis isused with enzymatic disruption methods (see below) for lysing bacterialcell walls. In addition, detergents are used to lyse and break downcellular membranes by solubilizing the lipids and membrane proteins onthe surface of cells. The contents of the cells include, in addition tothe desired nucleic acids, inner cellular proteins and cellular debris.Enzymes and other inhibitors are added after lysis to inactivatenucleases that may degrade the nucleic acids. Proteinase K is commonlyadded after lysis, destroying DNase and RNase enzymes capable ofdegrading the nucleic acids. After treatment with enzymes, the sample iscentrifuged, pelleting cellular debris, while the nucleic acids remainin the solution. The nucleic acids may be further purified as describedabove.

Another form of chemical lysis is the widely used procedure ofphenol-chloroform extraction. Phenol-chloroform extraction involves theability for nucleic acids to remain soluble in an aqueous solution in anacidic environment, while the proteins and cellular debris can bepelleted down via centrifugation. Phenol and chloroform ensure a clearseparation of the aqueous and organic (debris) phases. For DNA, a pH of7-8 is used, and for RNA, a more acidic pH of 4.5 is used.

Enzymatic lysis: Enzymatic disruption methods are commonly combined withother lysis methods such as those described above to disrupt cellularwalls (bacteria and plants) and membranes. Enzymes such as lysozyme,lysostaphin, zymolase, and protease are often used in combination withother techniques such as physical and chemical lysis. For example, onecan use cellulase to disrupt plant cell walls, lysosomes to disruptbacterial cell walls and zymolase to disrupt yeast cell walls.

Example II: RNP Formation

For RNP complex formation, 250 nM of LbCas12a nuclease protein wasincubated with 375 nM of a target specific gRNA in 1× Buffer (10 mMTris-HCl, 100 μg/mL BSA) with 2-15 mM MgCl₂ at 25° C. for 20 minutes.The total reaction volume was 2 μL. Other ratios of LbCas12a nuclease togRNAs were tested, including 1:1, 1:2 and 1:5. The incubationtemperature ranged from 16° C.-37° C., and the incubation time rangedfrom 10 minutes to 4 hours.

Example III: Blocked Nucleic Acid Molecule Formation

Ramp cooling: For formation of the secondary structure of blockednucleic acid molecules, 2.504 of a blocked nucleic acid molecule (any ofFormulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl)with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes todehybridize any secondary structures. Thereafter, the reaction wascooled to 37° C. at 0.015° C./second to form the desired secondarystructure.

Snap cooling: For formation of the secondary structure of blockednucleic acid molecules, 2.504 of a blocked nucleic acid molecule (any ofFormulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl)with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes todehybridize any secondary structures. Thereafter, the reaction wascooled to room temperature by removing the heat source to form thedesired secondary structure.

Snap cooling on ice: For formation of the secondary structure of blockednucleic acid molecules, 2.504 of a blocked nucleic acid molecule (any ofFormulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl)with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes todehybridize any secondary structures. Thereafter, the reaction wascooled to room temperature by placing the reaction tube on ice to formthe desired secondary structure.

Example IV: Reporter Moiety Formation

The reporter moieties used in the reactions herein were single-strandedDNA oligonucleotides 5-9 bases in length (e.g., with sequences of TTATT,TTTATTT, ATTAT, ATTTATTTA, AAAAA, or AAAAAAAAA) with a fluorophore and aquencher attached on the 5′ and 3′ ends, respectively. In one exampleusing a Cas12a cascade, the fluorophore was FAM-6 and the quencher wasIOWA BLACK® (Integrated DNA Technologies, Coralville, IA). In anotherexample using a Cas13 cascade, the reporter moieties weresingle-stranded RNA oligonucleotides 5-10 bases in length (e.g., r(U)n,r(UUAUU)n, r(A)n).

Example V: Cascade Assay

Format I (final reaction mix components added at the same time): RNP1was assembled using the LbCas12a nuclease and a gRNA for the Methicillinresistant Staphylococcus aureus (MRSA) DNA according to the RNP complexformation protocol described in Example II (for this sequence, seeExample VI). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nMof the MRSA-target specific gRNA. Next, RNP2 was formed using theLbCas12a nuclease and a gRNA specific for a selected blocked nucleicacid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembledwith 750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (New England Biolabs, Ipswich, MA) with 5 mM MgCl₂ at 25°C. for 20-40 minutes. Following incubation, RNP1s were diluted to aconcentration of 75 nM LbCas12a: 112.5 nM gRNA. Thereafter, the finalreaction was carried out in 1× Buffer, with 500 nM of the ssDNA reportermoiety, 1×ROX dye (Thermo Fisher Scientific, Waltham, MA) for passivereference, 2.5 mM MgCl₂, 4 mM NaCl, 15 nM LbCas12a: 22.5 nM gRNA RNP1,20 nM LbCas12a: 35 nM gRNA RNP2, and 50 nM blocked nucleic acid molecule(any one of Formula I-IV) in a total volume of 9 μL. 1 μL of MRSA DNAtarget (with samples having as low as three copies and as many as 30000copies—see FIGS. 6-14 ) was added to make a final volume of 10 μL. Thefinal reaction was incubated in a thermocycler at 25° C. withfluorescence measurements taken every 1 minute.

Format II (RNP1 and MRSA target pre-incubated before addition to finalreaction mix): RNP1 was assembled using the LbCas12a nuclease and a gRNAfor the MRSA DNA according to RNP formation protocol described inExample II (for this sequence, see Example VI). Briefly, 250 nM LbCas12anuclease was assembled with 375 nM of the MRSA-target specific gRNA.Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specificfor a selected blocked nucleic acid molecule (Formula I-IV) using 500 nMLbCas12a nuclease assembled with 750 nM of the blocked nucleicacid-specific gRNA incubated in 1×NEB 2.1 Buffer (New England Biolabs,Ipswich, MA) with 5 mM MgCl₂ at 25° C. for 20-40 minutes. Followingincubation, RNP1s were diluted to a concentration of 75 nM LbCas12a:112.5 nM gRNA. After dilution, the formed RNP1 was mixed with lilt ofMRSA DNA target and incubated at 16° C.-37° C. for up to 10 minutes toactivate RNP1. The final reaction was carried out in 1× Buffer, with 500nM of the ssDNA reporter moiety, 1×ROX dye (Thermo Fisher Scientific,Waltham, MA) for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, thepre-incubated and activated RNP1, LbCas12a: 35 nM gRNA RNP2, and 50 nMblocked nucleic acid molecule (any one of Formula I-IV) in a totalvolume of 9 μL. The final reaction was incubated in a thermocycler at25° C. with fluorescence measurements taken every 1 minute.

Format III (RNP1 and MRSA target pre-incubated before addition to finalreaction mix and blocked nucleic acid molecule added to final reactionmix last): RNP1 was assembled using the LbCas12a nuclease and a gRNA forthe MRSA DNA according to the RNP complex formation protocol describedin Example II (for this sequence, see Example VI). Briefly, 250 nMLbCas12a nuclease was assembled with 375 nM of the MRSA-target specificgRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNAspecific for a selected blocked nucleic acid molecule (Formula I-IV)using 500 nM LbCas12a nuclease assembled with 750 nM of the blockednucleic acid-specific gRNA incubated in 1× NEB 2.1 Buffer (New EnglandBiolabs, Ipswich, MA) with 5 mM MgCl₂ at 25° C. for 20-40 minutes.Following incubation, RNP1s were diluted to a concentration of 75 nMLbCas12a: 112.5 nM gRNA. After dilution, the formed RNP1 was mixed withlilt of MRSA DNA target and incubated at 16° C.-37° C. for up to 10minutes to activate RNP1. The final reaction was carried out in 1×Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (ThermoFisher Scientific, Waltham, MA) for passive reference, 2.5 mM MgCl₂, 4mM NaCl, the pre-incubated and activated RNP1, and LbCas12a: 35 nM gRNARNP2 in a total volume of 9 μL. Once the reaction mix was made, lilt (50nM) blocked nucleic acid molecule (any one of Formula I-IV) was addedfor a total volume of 10 μL. The final reaction was incubated in athermocycler at with fluorescence measurements taken every 1 minute.

Example VI: Detection of MRSA and Test Reaction Conditions

To detect the presence of Methicillin resistant Staphylococcus aureus(MRSA) and determine the sensitivity of detection with the cascadeassay, titration experiments with a MRSA DNA target nucleic acid ofinterest were performed. The MRSA DNA sequence (NCBI Reference SequenceNC: 0077911) is as follows.

SEQ ID NO: 615: ATGAAAAAGATAAAAATTGTTCCACTTATTTTAATAGTTGTAGTTGTCGGGTTTGGTATATATTTTTATGCTTCAAAAGATAAAGAAATTAATAATACTATTGATGCAATTGAAGATAAAAATTTCAAACAAGTTTATAAAGATAGCAGTTATATTTCTAAAAGCGATAATGGTGAAGTAGAAATGACTGAACGTCCGATAAAAATATATAATAGTTTAGGCGTTAAAGATATAAACATTCAGGATCGTAAAATAAAAAAAGTATCTAAAAATAAAAAACGAGTAGATGCTCAATATAAAATTAAAACAAACTACGGTAACATTGATCGCAACGTTCAATTTAATTTTGTTAAAGAAGATGGTATGTGGAAGTTAGATTGGGATCATAGCGTCATTATTCCAGGAATGCAGAAAGACCAAAGCATACATATTGAAAATTTAAAATCAGAACGTGGTAAAATTTTAGACCGAAACAATGTGGAATTGGCCAATACAGGAACAGCATATGAGATAGGCATCGTTCCAAAGAATGTATCTAAAAAAGATTATAAAGCAATCGCTAAAGAACTAAGTATTTCTGAAGACTATATCAAACAACAAATGGATCAAAATTGGGTACAAGATGATACCTTCGTTCCACTTAAAACCGTTAAAAAAATGGATGAATATTTAAGTGATTTCGCAAAAAAATTTCATCTTACAACTAATGAAACAGAAAGTCGTAACTATCCTCTAGGAAAAGCGACTTCACATCTATTAGGTTATGTTGGTCCCATTAACTCTGAAGAATTAAAACAAAAAGAATATAAAGGCTATAAAGATGATGCAGTTATTGGTAAAAAGGGACTCGAAAAACTTTACGATAAAAAGCTCCAACATGAAGATGGCTATCGTGTCACAATCGTTGACGATAATAGCAATACAATCGCACATACATTAATAGAGAAAAAGAAAAAAGATGGCAAAGATATTCAACTAACTATTGATGCTAAAGTTCAAAAGAGTATTTATAACAACATGAAAAATGATTATGGCTCAGGTACTGCTATCCACCCTCAAACAGGTGAATTATTAGCACTTGTAAGCACACCTTCATATGACGTCTATCCATTTATGTATGGCATGAGTAACGAAGAATATAATAAATTAACCGAAGATAAAAAAGAACCTCTGCTCAACAAGTTCCAGATTACAACTTCACCAGGTTCAACTCAAAAAATATTAACAGCAATGATTGGGTTAAATAACAAAACATTAGACGATAAAACAAGTTATAAAATCGATGGTAAAGGTTGGCAAAAAGATAAATCTTGGGGTGGTTACAACGTTACAAGATATGAAGTGGTAAATGGTAATATCGACTTAAAACAAGCAATAGAATCATCAGATAACATTTTCTTTGCTAGAGTAGCACTCGAATTAGGCAGTAAGAAATTTGAAAAAGGCATGAAAAAACTAGGTGTTGGTGAAGATATACCAAGTGATTATCCATTTTATAATGCTCAAATTTCAAACAAAAATTTAGATAATGAAATATTATTAGCTGATTCAGGTTACGGACAAGGTGAAATACTGATTAACCCAGTACAGATCCTTTCAATCTATAGCGCATTAGAAAATAATGGCAATATTAACGCACCTCACTTATTAAAAGACACGAAAAACAAAGTTTGGAAGAAAAATATTATTTCCAAAGAAAATATCAATCTATTAACTGATGGTATGCAACAAGTCGTAAATAAAACACATAAAGAAGATATTTATAGATCTTATGCAAACTTAATTGGCAAATCCGGTACTGCAGAACTCAAAATGAAACAAGGAGAAACTGGCAGACAAATTGGGTGGTTTATATCATATGATAAAGATAATCCAAACATGATGATGGCTATTAATGTTAAAGATGTACAAGATAAAGGAATGGCTAGCTACAATGCCAAAATCTCAGGTAAAGTGTATGATGAGCTATATGAGAACGGTAATAAAAAATACGATATAGA TGAATAA

Briefly, a RNP1 was preassembled with a gRNA sequence designed to targetMRSA DNA. Specifically, RNP1 was designed to target a 20 bp region ofthe mecA gene of MRSA: TGTATGGCATGAGTAACGAA (SEQ ID NO: 616). An RNP2was preassembled with a gRNA sequence designed to target the unblockednucleic acid molecule that results from unblocking (i.e., linearizing)blocked nucleic acid molecule U29 (FIG. 10A). The reaction mix containedthe preassembled RNP1, preassembled RNP2, and a blocked nucleic acidmolecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mMNaCl.

FIG. 10A shows the structure and segment parameters of molecule U29.Note molecule U29 has a secondary structure free energy value of −5.84kcal/mol and relatively short self-hybridizing, double-stranded regionsof 5 bases and 6 bases. FIGS. 10B-10H show the results achieved fordetection of 3E4 copies, 30 copies, 3 copies and copies of the mecA geneof MRSA (n=3) at 25° C. with varying concentrations of blocked nucleicacid, RNP2 and reporter moiety. FIG. 10B shows the results achieved when100 nM blocked nucleic acid molecules, 10 nM RNP2s and 500 nM reportermoieties are used. Thus, in this experiment, the ratio of blockednucleic acid molecules to RNP2s is 10:1. Note first that with 3E4copies, nearly 100% of the reporters are cleaved at t=0 with asignal-to-noise ratio of 28.06 at 0 minutes, a signal-to-noise ratio of24.23 at 5 minutes, and a signal-to-noise ratio of 21.01 at 10 minutes.Additionally, the signal-to-noise ratios for detection with 30 copies ofMRSA target is 12.45 at 0 minutes, 14.07 at 5 minutes and 16.16 at 10minutes; and the signal-to-noise ratios for detection with 3 copies ofMRSA target is 1.79 at 0 minutes, 1.64 at 5 minutes and is 2.04 at 10minutes. Note the measured fluorescence at 0 copies increases onlyslightly over the 10- and 30-minutes intervals, resulting in a flatnegative. A flat negative (the results obtained over the time period for0 copies) demonstrates that there is very little non-specific orundesired signal generation in the system. Note that the negative whenthe ratio of blocked nucleic acid molecules to RNP2s is 10:1 is flatterthan those in FIGS. 10C through 10H.

FIG. 10C shows the results achieved when 50 nM blocked nucleic acidmolecules, 10 nM RNP2s and 500 nM reporter moieties are used. Thus, inthis experiment, the ratio of blocked nucleic acid molecules to RNP2s is5:1. Note first that with 3E4 copies, again nearly 100% of the reportersare cleaved at t=0 with a signal-to-noise ratio of 12.85, asignal-to-noise ratio of 10.51 at 5 minutes, and a signal-to-noise ratioof 8.18 at 10 minutes. Additionally, the signal-to-noise ratios fordetection with 30 copies of MRSA target is 5.85 at 0 minutes, 6.44 at 5minutes and 6.48 at 10 minutes; and the signal-to-noise ratios fordetection with 3 copies of MRSA target is 1.54 at 0 minutes, 1.61 at 5minutes and is 1.71 at 10 minutes. Note the measured fluorescence at 0copies increases, resulting in less of a flat negative than the 10:1ratio of blocked nucleic acid molecules to RNP2.

FIG. 10D shows the results achieved when 50 nM blocked nucleic acidmolecules, 10 nM RNP2s and 2500 nM reporter moieties are used. Thus, inthis experiment, the ratio of blocked nucleic acid molecules to RNP2s is5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved att=0 with a signal-to-noise ratio of 34.92, a signal-to-noise ratio of30.62 at 5 minutes, and a signal-to-noise ratio of 25.81 at 10 minutes.Additionally, the signal-to-noise ratios for detection with 30 copies ofMRSA target is 7.97 at 0 minutes, 1.73 at 5 minutes and 10.50 at 10minutes; and the signal-to-noise ratios for detection with 3 copies ofMRSA target is 1.65 at 0 minutes, 1.73 at 5 minutes and is 1.82 at 10minutes. Note the measured fluorescence at 0 copies increases, resultingin less of a flat negative than the 10:1 ratio of blocked nucleic acidmolecules to RNP2s, but likely due to the 5× increase in theconcentration of reporter moieties; however, note also that a higherconcentration of reporter moieties allows for a higher signal-to-noiseratio for 3E4 and 30 copies of MRSA target.

FIG. 10E shows the results achieved when 100 nM blocked nucleic acidmolecules, 20 nM RNP2s and 500 nM reporter moieties are used and 4 mMNaCl. Thus, in this experiment, the ratio of blocked nucleic acidmolecules to RNP2s is 5:1 but double the concentration of both of thesemolecules than that shown in FIGS. 10C and 10D. With 3E4 copies, againnearly 100% of the reporters are cleaved at t=0 with a signal-to-noiseratio of 11.89, a signal-to-noise ratio of 8.97 at 5 minutes, and asignal-to-noise ratio of 6.53 at 10 minutes. Additionally, thesignal-to-noise ratios for detection with 30 copies of MRSA target is5.46 at 0 minutes, 5.85 at 5 minutes and 5.43 at 10 minutes; and thesignal-to-noise ratios for detection with 3 copies of MRSA target is1.58 at 0 minutes, 1.65 at 5 minutes and is 1.80 at 10 minutes. Note themeasured fluorescence at 0 copies increases, resulting in less of a flatnegative than the 10:1 ratio of blocked nucleic acid molecules to RNP2sshown in FIG. 10B. Note also that the ratio of blocked nucleic acidmolecules to RNP2s (5:1) appears to be more important than the ultimateconcentration (100 nM/20 nM) by comparison to FIG. 10D where the ratioof blocked nucleic acid molecules to RNP2s was also 5:1 however theconcentration of blocked nucleic acid molecules was 50 nM and theconcentration of RNP2 was 10 nM.

FIG. 10F shows the results achieved when 50 nM blocked nucleic acidmolecules, 20 nM RNP2s and 500 nM reporter moieties are used and using aconcentration of 4 mM NaCl. In this experiment the ratio of blockednucleic acid molecules to RNP2s is 2.5:1. With 3E4 copies, again nearly100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of25.85, a signal-to-noise ratio of 21.36 at 5 minutes, and asignal-to-noise ratio of 16.24 at 10 minutes. Additionally, thesignal-to-noise ratios for detection with 30 copies of MRSA target is5.28 at 0 minutes, 6.19 at 5 minutes and 7.02 at 10 minutes; and thesignal-to-noise ratios for detection with 3 copies of MRSA target isvery low at 0 minutes, 1.53 at 5 minutes and is 1.73 at 10 minutes. Notethe measured fluorescence at 0 copies increases, resulting in less of aflat negative than the 10:1 ratio of blocked nucleic acid molecules toRNP2s shown in FIG. 10B. Note also that the signal-to-noise ratio forall concentrations was reduced at the 2.5:1 ratio of blocked nucleicacid molecules to RNP2s.

FIG. 10G shows the results achieved when 50 nM blocked nucleic acidmolecules, 20 nM RNP2s and 500 nM reporter moieties are used and using aconcentration of 10 mM NaCl. Thus, in this experiment, the ratio ofblocked nucleic acid molecules to RNP2s is 2.5:1. With 3E4 copies, againnearly 100% of the reporters are cleaved at t=0 with a signal-to-noiseratio of 12.75, a signal-to-noise ratio of 7.78 at 5 minutes, and asignal-to-noise ratio of 3.66 at 10 minutes. Additionally, thesignal-to-noise ratios for detection with 30 copies of MRSA target is6.09 at 0 minutes, 6.23 at 5 minutes and 3.58 at 10 minutes; and thesignal-to-noise ratios for detection with 3 copies of MRSA target isvery low at 0 minutes, 1.40 at 5 minutes and is 1.62 at 10 minutes. Notethe measured fluorescence at 0 copies increases, resulting in less of aflat negative than the 10:1 ratio of blocked nucleic acid molecules toRNP2s shown in FIG. 10B. Note also that the signal-to-noise ratio forall concentrations was reduced substantially at the 2.5:1 ratio ofblocked nucleic acid molecules to RNP2s and that the NaCl concentrationat 10 mM vs. 4 mM (FIG. 10F) did not make much of a difference.

FIG. 10H shows the results achieved when 100 nM blocked nucleic acidmolecules, 20 nM RNP2s and 500 nM reporter moieties are used and using aconcentration of 10 mM NaCl. Thus, in this experiment, the ratio ofblocked nucleic acid molecules to RNP2s is 5:1. With 3E4 copies, againnearly 100% of the reporters are cleaved at t=0 with a signal-to-noiseratio of 77.38, a signal-to-noise ratio of 74.18 at 5 minutes, and asignal-to-noise ratio of 67.90 at 10 minutes. Additionally, thesignal-to-noise ratios for detection with 30 copies of MRSA target is5.94 at 0 minutes, 7,45 at 5 minutes and 9.73 at 10 minutes; and thesignal-to-noise ratios for detection with 3 copies of MRSA target is1.66 at 0 minutes, 2.13 at 5 minutes and is 2.38 at 10 minutes. Note themeasured fluorescence at 0 copies increases slightly, resulting in lessof a flat negative than the 10:1 ratio of blocked nucleic acid moleculesto RNP2s shown in FIG. 10B. Note also that the signal-to-noise ratio forall concentrations was increased substantially at the 5:1 ratio ofblocked nucleic acid molecules to RNP2s as compared to the 2.5:1 rationof blocked nucleic acid molecules to RNP2s. In summary, the resultsshown in FIGS. 10B-10H indicate that a 5:1 ratio of blocked nucleic acidmolecules to RNP2s or greater leads to higher signal-to-noise ratios forall concentrations of MRSA target.

Example VII: Homology Modeling and Mutation Structure Analysis

The variant nucleic acid-guided nucleases presented herein weredeveloped in the following manner: For protein engineering and aminoacid substitution model predictions, a first Protein Data Bank (pdb)file with the amino acid sequence and structure information for the RNPcomprising the base nucleic acid-guided nuclease to be mutated, the gRNAand a bound dsDNA target nucleic acid was obtained. (For structuralinformation for RNPs comprising AsCas12s and LbCas12a, see, e.g.,Yamano, et al., Molecular Cell, 67:633-45 (2017).) Desired and/or randomamino acid substitutions were then “made” to the base nucleicacid-guided nuclease (LbCas12a), the resulting structural change to thebase nucleic acid-guided nuclease due to each amino acid substitutionwas used to generate updated files for the resulting RNPs comprisingeach of the variant nucleic acid-guided nucleases using SWISS-MODEL andthe original pdf file as a reference template. SWISS-MODEL worked wellin the present case as the amino acid sequences of wildtype LbCas12a wasknown, as were the planned amino acid substitutions. The output of theupdated files for each variant nucleic acid-guided nuclease included aroot mean square deviation (RMSD) value for the structural changescompared to the RNP complex for wt LbCas12a in Angstrom units (i.e., ameasurement of the difference between the backbones of wt LbCas12a andthe variant nucleic acid-guided nuclease) and the updated pdb files ofthe variant nucleic acid-guided nucleases are further assessed at thepoint of mutations for changes in the hydrogen bonds compared to thereference original pdb file of the nuclease.

After SWISS modeling, an independent step for calculating free energywas performed using, e.g., a Flex ddG module based on the programRosetta CM to extract locally destabilizing mutations. This was used asa proxy for amino acid interference with PAM regions of the DNA toassess the probability of unwinding of the target nucleic acid. (See,e.g., Shanthirabalan, et al., Proteins: Structure, Function, andBioinformatics 86(8):853-867 (2018); and Barlow, et al., J. PhysicalChemistry B, 122(21):5389-99 (2018).)

Generally, the results of the SWISS-Model and Rosetta analysis indicatedthat stable enzyme function related to the PAM domain would require aglobal RMSD value range from 0.1 to 2.1 angstroms, and the following ΔΔGFlex Values: for stabilizing mutations ΔΔG≤−1.0 kcal/mol; for neutralmutations: −1.0 kcal/mol<ΔΔG<1.0 kcal/mol; and for destabilizingmutations: ΔΔG≥1.0 kcal/mol. Sixteen single mutations were identifiedthat, singly or in combination, met the calculated criteria. Structuralmodeling for mutations at four exemplary amino acid residues aredescribed below.

FIG. 6A shows the result of protein structure prediction using Rosettaand SWISS modeling of wildtype LbCas12a (Lachnospriaceae bacteriumCas12a). Protein structure prediction using Rossetta and SWISS modelingof exemplary variants of wildtype LbCas12a are shown below.

Mutation 1, G532A: The structure of an RNP comprising the G532A variantnucleic acid-guided nuclease is shown in FIG. 11A. Modeling indicatedthe following changes to the wildtype LbCas12a structure with the G532Asubstitution (seen in FIG. 11A as a red residue): loss of one hydrogenbond with TS-PAM (target strand PAM) at amino acid residue 595; loss ofone hydrogen bond with NTS-PAM (non-target strand PAM) at amino acidresidue 595; no addition or loss of a hydrogen bond at amino acidresidue 532. Per simulations, mutation G532A is a structurallystabilizing mutation. The parameters collected from SWISS-MODEL andRosetta analysis are shown in Table 17.

TABLE 17 Mutation 1: G532A Global RMSD: 0.976 PI RMSD: 0.361 REC1 RMSD:0.289 (235 to 235 atoms) WED RMSD: 0.306 (198 to 198 atoms) ΔΔG FlexValue: −1.13 PI = PAM-interacting domain of the G532A variant REC1 =REC1 domain of the G532A variant WED = WED domain of the G532A variant

Mutation 2, K538A: The structure of an RNP comprising the K538A variantnucleic acid-guided nuclease is shown at left in FIG. 11B. Modelingindicated the following changes to the wildtype LbCas12a structure withthe K538A substitution (seen in FIG. 11B as a pink residue): loss of onehydrogen bond with TS-PAM (target strand PAM) at amino acid residue 538;loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acidresidue 595; loss of one hydrogen bond with NTS-PAM (non-target strandPAM) at amino acid residue 595. Per simulations, mutation K538A is astructurally stabilizing mutation. The parameters collected fromSWISS-MODEL and Rosetta analysis are shown in Table 18.

TABLE 18 Mutation 2: K538A Global RMSD: 0.990 PI RMSD: 0.376 REC1 RMSD:0.305 (236 to 236 atoms) WED RMSD: 0.324 (194 to 194 atoms) ΔΔG FlexValue: 0.06 PI = PAM-interacting domain of the K538A variant REC1 = REC1domain of the K538A variant WED = WED domain of the K538A variant

Mutation 3, Y542A: The structure of an RNP comprising the Y542A variantnucleic acid-guided nuclease is shown in FIG. 11C. Modeling indicatedthe following changes to the wildtype LbCas12a structure with the Y542Asubstitution (seen in FIG. 11C as a blue residue): loss of two hydrogenbonds with TS-PAM (target strand PAM) at amino acid residue 542; loss ofone hydrogen bond with TS-PAM (target strand PAM) at amino acid residue538; loss of one hydrogen bond with TS-PAM (target strand PAM) at aminoacid residue 595; loss of one hydrogen bond with NTS-PAM (non-targetstrand PAM) at amino acid residue 595. Per simulations, mutation Y542Ais a structurally stabilizing mutation. The parameters collected fromSWISS-MODEL and Rosetta analysis are shown in Table 19.

TABLE 19 Mutation 3: Y542A Global RMSD: 0.989 PI RMSD: 0.377 REC1 RMSD:0.306 (237 to 237 atoms) WED RMSD: 0.338 (199 to 199 atoms) ΔΔG FlexValue: −2.06 PI = PAM-interacting domain of the Y542A variant REC1 =REC1 domain of the Y542A variant WED = WED domain of the Y542A variant

Mutation 4, K595A: The structure of an RNP comprising the K595A variantnucleic acid-guided nuclease is shown in FIG. 11D. Modeling indicatedthe following changes to the wildtype LbCas12a structure with the K595Asubstitution (seen in FIG. 11D as an orange residue): loss of twohydrogen bonds with TS-PAM (target strand PAM) at amino acid residue595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) atamino acid residue 595; loss of one hydrogen bond with NTS-PAM(non-target strand PAM) at amino acid residue 538. Per simulations,mutation K595A is a structurally destabilizing mutation. The parameterscollected from SWISS-MODEL and Rosetta analysis are shown in Table 20.

TABLE 20 Mutation 4: K595A Global RMSD: 0.976 PI RMSD: 0.361 REC1 RMSD:0.289 (235 to 235 atoms) WED RMSD: 0.306 (198 to 198 atoms) ΔΔG FlexValue: 1.26 PI = PAM-interacting domain of the K595A variant REC1 = REC1domain of the K595A variant WED = WED domain of the K595A variant

Mutation 5, Combination G532A, K538A, Y542A, and K595A: The structure ofan RNP comprising the combination G532A/K538A/Y542A/K595A variant(“combination variant”) nucleic acid-guided nuclease is shown in FIG.11E. Modeling indicated the following changes to the wildtype LbCas12astructure with the four substitutions: loss of five hydrogen bonds withTS-PAM (target strand PAM); loss of one hydrogen bond with NTS-PAM(non-target strand PAM). Per simulations, the combination variant isstructurally stable. The parameters collected from SWISS-MODEL andRosetta analysis are shown in Table 21.

TABLE 21 Mutation 5: G532A/K538A/Y542A/K595A Global RMSD: 0.966 PI RMSD:0.351 REC1 RMSD: 0.261 (226 to 226 atoms) WED RMSD: 0.288 (200 to 200atoms) ΔΔG Flex Value: −3.31 PI = PAM-interacting domain of thecombination variant REC1 = REC1 domain of the combination variant WED =WED domain of the combination variant

Mutation 6, K595D: The structure of an RNP comprising the K595D variantnucleic acid-guided nuclease is shown in FIG. 11F. Modeling indicatedthe following changes to the wildtype LbCas12a structure at location 595with this substitution: loss of two hydrogen bonds with TS-PAM (targetstrand PAM); loss of one hydrogen bond with NTS-PAM (non-target strandPAM); and gain of one hydrogen bond with NTS-PAM. Per simulations, theK595D variant is structurally unstable. The parameters collected fromSWISS-MODEL and Rosetta analysis are shown in Table 22.

TABLE 22 Mutation 6: K595D Global RMSD: 1.001 PI RMSD: 0.367 (89 to 89atoms) REC1 RMSD: 0.296 (235 to 235 atoms) WED RMSD: 0.320 (197 to 197atoms) ΔΔG Flex Value: 2.04 PI = PAM-interacting domain of thecombination variant REC1 = REC1 domain of the combination variant WED =WED domain of the combination variant

Mutation 7, K595E: The structure of an RNP comprising the K595E variantnucleic acid-guided nuclease is shown in FIG. 11G. Modeling indicatedthe following changes to the wildtype LbCas12a structure at location 595with this substitution: loss of two hydrogen bonds with TS-PAM (targetstrand PAM); loss of one hydrogen bond with NTS; and no gain of hydrogenbonds. Per simulations, the K595E variant is structurally unstable. Theparameters collected from SWISS-MODEL and Rosetta analysis are shown inTable 23.

TABLE 23 Mutation 6: K595E Global RMSD: 0.975 PI RMSD: 0.352 (89 to 89atoms) REC1 RMSD: 0.264 (226 to 226 atoms) WED RMSD: 0.290 (198 to 198atoms) ΔΔG Flex Value: 1.37 PI = PAM-interacting domain of thecombination variant REC1 = REC1 domain of the combination variant WED =WED domain of the combination variant

Mutation 8, Combination K538A, Y542A, K595D: The structure of an RNPcomprising the combination K538A/Y542A/K595D variant (“combinationvariant”) nucleic acid-guided nuclease is shown in FIG. 11H. Modelingindicated the following changes to the wildtype LbCas12a structure withthe three substitutions: loss of two hydrogen bonds with TS (targetstrand) at position 595; loss of one hydrogen bond with NTS(non-target); combined loss of three hydrogen bonds at 532/242positions; and gain of one hydrogen bond at 595. Per simulations, thecombination variant is structurally destabilizing. The parameterscollected from SWISS-MODEL and Rosetta analysis are shown in Table 24.

TABLE 24 Mutation 6: K538A, Y542A, K595D Global RMSD: 0.976 PI RMSD:0.351 (89 to 89 atoms) REC1 RMSD: 0.261 (225 to 225 atoms) WED RMSD:0.289 (198 to 198 atoms) ΔΔG Flex Value: 0.96 PI = PAM-interactingdomain of the combination variant REC1 = REC1 domain of the combinationvariant WED = WED domain of the combination variant

Mutation 9, Combination K538A, Y542A, K595E: The structure of an RNPcomprising the combination K538A/Y542A/K595E variant (“combinationvariant”) nucleic acid-guided nuclease is shown in FIG. 11I. Modelingindicated the following changes to the wildtype LbCas12a structure withthe three substitutions: loss of two hydrogen bonds with TS (targetstrand) at position 595; loss of one hydrogen bond with NTS(non-target); combined loss of three hydrogen bonds at 532/242positions. Per simulations, the combination variant is structurallystabilizing. The parameters collected from SWISS-MODEL and Rosettaanalysis are shown in Table 25.

TABLE 25 Mutation 6: K538A, Y542A, K595E Global RMSD: 0.976 PI RMSD:0.351 (89 to 89 atoms) REC1 RMSD: 0.261 (225 to 225 atoms) WED RMSD:0.289 (198 to 198 atoms) ΔΔG Flex Value: −3.71 PI = PAM-interactingdomain of the combination variant REC1 = REC1 domain of the combinationvariant WED = WED domain of the combination variant

In addition to amino acid substitutions, modifications, such as chemicalmodifications, can be made to amino acids identified by the structuraland homology modeling described above. FIG. 6G illustrates an exemplaryscheme for acetylating amino acid residue 595 in LbCas12a, amodification which prevents unwinding of dsDNA by blocking entry of atarget nucleic acid into the RNP via steric hindrance. LbCas12a iscombined with AcrVA5 and the reaction is incubated for 20 minutes atroom temperature, resulting in LECas12a that has been acetylated atamino acid residue 595 (K595K Ac). (For a discussion and methods fordisabling of Cas12a by ArVA5, see Dong, et al., Nature Structural andMolecular Bio., 26(4):308-14 (2019).) DsDNA is not a substrate forLbCas12a with a K595K Ac modification; however, ssDNA is a substrate forLbCas12a with a K595K Ac modification; thus, LbCas12a (K595K Ac) has thedesired properties of the variant nucleic acid-guided nucleasesdescribed above. In addition to acetylation, phosphorylation andmethylation of select amino acid residues may be employed.

Example VIII: Single-strand Specificity of the Variant NucleicAcid-Guided Nucleases

In vitro transcription/translation reactions were performed for variantLbaCas12a nucleases as noted in Table 26 using the nucleic acidsequences listed in Table 27:

TABLE 26 Template DNA for  250 ng IVTT gRNA concentration 100 nMDNA activator   25 nM concentration Probe concentration 500 nMReaction volume  30 UL Reporter 5′-FAM-TTATTATT-IABKFQ-3′ PlatePCR plate 96-well, black Read temperature  25° C. Read duration 30 minutes Buffer NEB r2.1 New England  Biolabs ®, Inc., Ipswich,  MA)Na+  50 mM Mg + 2  10 mM

TABLE 27 Activator RunX  GCCTTCAGAAGAGGGTGCAT fragmentTTTCAGGAGGAAGCGATGGC (dsDNA +  TTCAGACAGCATATTTGAGT PAM) CATT (SEQ ID NO. 617) RunX  GCCTTCAGAAGAGGGTGCAT fragmentGCACAGGAGGAAGCGATGGC (dsDNA -  TTCAGACAGCATATTTGAGT PAM) CATT (SEQ ID NO. 618) Target  AGGAGGAAGCGATGGCTTCAGA  region in(SEQ ID NO. 619) activator gRNA LbaCas  gUAAUUUCUACUAAGUGUAGAUAGGAGGAAGCGAUGGCUUCAGA 12a gRNA (SEQ ID NO. 620)

The results are shown in FIGS. 12A-12G indicating the time for detectionof dsDNA and ssDNA both with and without PAM sequences for purifiedwildtype LbaCas12a and three variants (K538A+K595A, K595A, andK538A+Y542+K595A, and unpurified engineered variants of LbaCas12a:K538D+Y542A+K595D, K595D, K538A+K595D, K538A+K595E,G532A+K538A+Y542A+K595A, K538A+Y542A+K595D, K538D+Y542A+K595A,K538D+Y542D+K595A, and K538E+Y542A+K595A. Note that all variantengineered nucleic acid-guided nucleases slowed down double-strand DNAdetection to varying degrees, with the double and triple variants atpositions K538, Y542 and K595 of wt LbaCas12a performing best incomparison to wt LbCas12a, while single-strand DNA detection remainedhigh, both in single-strand DNA with a PAM and without a PAM. Thefollowing variants were particularly robust: K538D+Y542A+K595D,K538A+K595D, K538A+K595E, G532A+K538A+Y542A+K595A, K538D+Y542A+K595A,and K538D+Y542D+K595D.

FIGS. 13A and 13B show the sequence alignment of many different Cas12anucleases and orthologs, including in some instances several alignmentsof the same Cas12a nuclease.

Example IX: Detection of Biomarker Alpha-Synuclein in CSF for MonitoringProgression of Parkinson's Disease

The biomarker α-synuclein, which is found in both aggregated andfibrillar form, has attracted attention as a biomarker of Parkinson'sdisease. Human α-synuclein is expressed in the brain in the neocortex,hippocampus, substantia nigra, thalamus and cerebellum. It is encoded bythe SNCA gene that consists of six exons ranging in size from 42 to 1110base pairs. The predominant form of α-synuclein is the full-lengthprotein, but other shorter isoforms exist. C-terminal truncation ofα-synuclein induces aggregation, suggesting that C-terminalmodifications may be involved in Parkinson's pathology. Changes in thelevels of α-synuclein have been reported in CSF of Parkinson' patients.The gradual spread of α-synuclein pathology leads to a highconcentration of extracellular α-synuclein that can potentially damagehealthy neurons. Here, the cascade assay is used to monitor the level ofnucleic acids in cerebrospinal fluid (CSF) to monitor the levels of mRNAtranscripts that when translated lead to a truncated α-synucleinprotein.

A lumbar puncture is performed on an individual, withdrawingapproximately 5 mL of cerebrospinal fluid (CSF) for testing. The CSFsample is then treated by phenol-chloroform extraction or oligo dTaffinity resins via a commercial kit (see, e.g., the TurboCapture mRNAkit or RNeaxy Pure mRNA Bead Kit from Qiagen®). Briefly, two RNP1s arepreassembled as described above in Example II with a first gRNA sequencedesigned to target the coding sequence of the mRNA transcribed from SNCAgene specific to the C-terminus region of α-synuclein to detectfull-length α-synuclein and second gRNA sequence designed to target thecoding sequence of the mRNA transcribed from SNCA gene specific to theN-terminus region of α-synuclein to detect all α-synuclein mRNAs. Inaddition to the gRNA, each RNP1 also comprises an LbCas13a nuclease(i.e., an RNA-specific nuclease). Also as described in Example II above,an RNP2 is preassembled with a gRNA sequence designed to target anunblocked nucleic acid molecule that results from unblocking (i.e.,linearizing) a chosen blocked nucleic acid molecule such as U29. Theblocked nucleic acid molecule is formed as described above in ExampleIII, and a reporter is formed as described above in Example IV. Thereaction mix contains the preassembled RNP1, preassembled RNP2, and ablocked nucleic acid molecule, in a buffer (pH of about 8) containing 4mM MgCl₂ and 101 mM NaCl. The cascade assay is performed by one of theprotocols described above in Example V. A readout is performed bycomparing the level of N-terminus coding sequences detected (the levelof total α-synuclein mRNA) versus the level of C-terminus codingsequences detected (the level of full-length α-synuclein mRNA).

Example X: Detection of Foot and Mouth Disease Virus from Nasal Swabs

Foot-and-mouth disease (FMD) is a severe and highly contagious viraldisease. The FMD virus causes illness in cows, pigs, sheep, goats, deer,and other animals with divided hooves and is a worldwide concern as itcan spread quickly and cause significant economic losses. FMD hasserious impacts on the livestock trade—a single detection of FMD willstop international trade completely for a period of time. Since thedisease can spread widely and rapidly and has grave economicconsequences, FMD is one of the animal diseases livestock owners dreadmost. FMD is caused by a virus, which survives in living tissue and inthe breath, saliva, urine, and other excretions of infected animals. FMDcan also survive in contaminated materials and the environment forseveral months under the right conditions.

A nasal swab is performed on a subject, such as a cow or pig, and thenucleic acids extracted using, e.g., the Monarch Total RNA Miniprep Kit(New England Biolabs®, Inc., Ipswich, MA). Briefly, an RNP1 ispreassembled as described above in Example II with a gRNA sequencedesigned to a gene from the FMD virus (e.g., to a portion of NCBIReference Sequence NC 039210.1) and an LbCas12a nuclease (i.e., aDNA-specific nuclease). Also as described in Example II above, an RNP2is preassembled with a gRNA sequence designed to target an unblockednucleic acid molecule that results from unblocking (i.e., linearizing) achosen blocked nucleic acid molecule such as U29. The blocked nucleicacid molecule is formed as described above in Example III, and areporter is formed as described above in Example IV. The reaction mixcontains the preassembled RNP1, preassembled RNP2, and a blocked nucleicacid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101mM NaCl. The cascade assay is performed by one of the protocolsdescribed above in Example V, and the readout is positive detection ofFMD virus-specific DNA sequences.

Example XI: Detection of Sickle Cell Gene Sequences in Peripheral Blood

Sickle cell disease (SCD) is a group of inherited red blood celldisorders. In someone who has SCD, the hemoglobin is abnormal, whichcauses the red blood cells to become hard and sticky and look like aC-shaped farm tool called a “sickle.” The sickle cells die early, whichcauses a constant shortage of red blood cells; in addition, when thesickle-shaped blood cells travel through small blood vessels, they getstuck and clog the blood flow, causing pain and other seriouscomplications such as infection and stroke.

One form of SCD is HbSS. Individuals who have this form of SCD inherittwo genes, one from each parent, that code for hemoglobin “S.”Hemoglobin S is an abnormal form of hemoglobin that causes the red cellsto become rigid and sickle shaped. This is commonly called sickle cellanemia and is usually the most severe form of the disease. Another formof SCD is HbSC. Individuals who have this form of SCD inherit ahemoglobin “S” gene from one parent and a gene for a different type ofabnormal hemoglobin called “C” from the other parent. This is usually amilder form of SCD. A third form of SCD is HbS thalassemia. Individualswho have this form of SCD inherit a hemoglobin “S” gene from one parentand a gene for beta thalassemia, another type of hemoglobin abnormality,from the other parent. There are two types of beta thalassemia: “zero”(HbS beta0) and “plus” (HbS beta+). Those with HbS beta0-thalassemiausually have a severe form of SCD. People with HbS beta+-thalassemiatend to have a milder form of SCD.

A non-invasive prenatal test (NIPT) that uses only maternal cell-freeDNA (cfDNA) from peripheral blood permits prenatal detection of sicklecell disease and beta thalassemia by screening without the need forpaternal DNA. Such a screening enables patients and healthcare providersto make informed decisions about diagnostic testing and may expand genetherapy treatment options. A 10 mL peripheral blood draw is performed ona pregnant subject into a Streck tube. The blood is treated withlysis-binding buffer and proteinase K under denaturing conditions at 55°C. for 15 minutes in the presence of magnetic beads. Following theheating step, the mixture is incubated for 1 hour at room temperaturewith mixing every 10 minutes at 1200 rpm for 30 seconds on an Eppendorfthemomixer. The beads are captured on a magnetic stand for 2 minutes,washed three times after which cfDNA is eluted by adding elution bufferand incubating for 5 minutes at 55° C. The cfDNA is further purified bydiluting in 1:1 FTA (Fast Technology for Analysis) reagent, cat#WHAWB120204 (Sigma-Aldrich, USA), containing NaCl (sodium chloride);Tris; EDTA (ethylenediaminetetraacetic acid); TRITON-X-100(t-Octylphenoxypolyethoxyethanol) and incubated for 10 minutes at roomtemperature. An additional bead purification step is performed usingPCRClean DX beads, cat #C-1003-450 (ALINE Biosciences, USA).Alternatively, there are several kits available commercially that aredesigned to extract cfDNA including the BioChain® cfPure® Cell free DNAExtraction Kit (BioChain®, Newark, CA); the Monarch Genomic DNAPurification Kit and the Monarch HMW DNA Extraction Kit for Blood (NewEngland Biolabs®, Inc., Ipswich, MA); and the cfDNA Purification Kit(Active Motif®, Carlsbad, CA).

For the cascade assay, three RNP1s are preassembled as described abovein Example II with 1) gRNA sequence designed to detect the Hemoglobin Sgene variant and an LbCas12a nuclease (i.e., an DNA-specific nuclease);2) a gRNA sequence designed to detect the Hemoglobin C gene variant andan LbCas 12a nuclease (i.e., an DNA-specific nuclease); and 3) a gRNAsequence designed to detect the gene for beta thalassemia and anLbCas12a nuclease (i.e., an DNA-specific nuclease). Also as described inExample II above, an RNP2 is preassembled with a gRNA sequence designedto target an unblocked nucleic acid molecule that results fromunblocking (i.e., linearizing) a chosen blocked nucleic acid moleculesuch as U29. The blocked nucleic acid molecule is formed as describedabove in Example III, and a reporter is formed as described above inExample IV. The reaction mix contains the preassembled RNP1,preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pHof about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay isperformed by one of the protocols described above in Example V. Thereadout is detection of the Hemoglobin S gene variant, the detection ofthe Hemoglobin S variant and the Hemoglobin C variant, and the detectionof the Hemoglobin S variant and the β-thalassemia gene.

Example XII: Detection of Donor-Derived Gene Sequences in PeripheralBlood of Transplant Patients

Costly and invasive tissue biopsies to detect allograft rejection aftertransplantation have numerous limitations; however, assays based oncell-free DNA (cfDNA)—circulating fragments of DNA released from cells,tissues, and organs as they undergo natural cell death—can improve theability to detect rejection and implement earlier changes in managementof the transplanted organ. Rejection, referring to injury of a donatedorgan caused by the recipient's immune system, often causes allograftdysfunction and even patient death. T-cell mediated acute cellularrejection occurs most often within the first 6 months post-transplant.Acute cellular rejection involves accumulation of CD4+ and CD8+ T-cellsin the interstitial space of the allograft as the recipient's immunesystem recognizes antigens on the donated organ as foreign, initiatingan immune cascade that ultimately leads to apoptosis of the targetedcells. As these cells die, genomic DNA is cleaved and fragments of donorderived-cfDNA are released to join the pool of recipient cfDNA in theblood. Using cfDNA as a biomarker for acute cellular rejection isadvantageous since it is derived from the injured cells of the donatedorgan and therefore should represent a direct measure of cell deathoccurring in the allograft. Further, cfDNA maintains all of the geneticfeatures of the original genomic DNA, allowing the genetic materialreleased from the donated organ to be differentiated from the cfDNAderived from cells of the recipient that are undergoing naturalapoptosis.

For organ transplants in which the donor is male and the recipient isfemale, this “sex mismatch” is leveraged to calculate donorderived-cfDNA levels from within the recipient's total cfDNA pool.Although this approach allows for confident diagnosis of rejection inthe allograft, sex-mismatch between the donor and recipient isrelatively infrequent and not universally applicable; thus, the presenceof other genetic differences between the donor and recipient at aparticular locus are leveraged to identify the origin of the circulatingcfDNA. Ideally, the recipient would be homozygous for a single base (forexample, AA) and at the same locus the donor would be homozygous for adifferent base (for example, GG). Given the genetic heterogeneitybetween individuals, hundreds to tens of thousands of potentiallyinformative loci across the genome can be interrogated to distinguishdonor derived-cfDNA from recipient cfDNA.

A 10 mL peripheral blood draw is performed on a transplantation subjectinto a Streck tube. The blood is treated with lysis-binding buffer andproteinase K under denaturing conditions at 55° C. for 15 minutes in thepresence of magnetic beads. Following the heating step, the mixture isincubated for 1 hour at room temperature with mixing every 10 minutes at1200 rpm for 30 seconds on an Eppendorf themomixer. The beads arecaptured on a magnetic stand for 2 minutes, washed three times afterwhich cfDNA is eluted by adding elution buffer and incubating for 5minutes at 55° C. The cfDNA is further purified by diluting in 1:1 FTA(Fast Technology for Analysis) reagent, cat #WHAWB120204 (Sigma-Aldrich,USA), containing NaCl (sodium chloride); Tris; EDTA(ethylenediaminetetraacetic acid); TRITON-X-100(t-Octylphenoxypolyethoxyethanol) and incubated for 10 minutes at roomtemperature. An additional bead purification step is performed usingPCRClean DX beads, cat #C-1003-450 (ALINE Biosciences, USA). Also, asstated above, there are several kits available commercially that aredesigned to extract cfDNA including the BioChain® cfPure® Cell free DNAExtraction Kit (BioChain®, Newark, CA); the Monarch Genomic DNAPurification Kit and the Monarch HMW DNA Extraction Kit for Blood (NewEngland Biolabs®, Inc., Ipswich, MA); and the cfDNA Purification Kit(Active Motif®, Carlsbad, CA).

For the cascade assay, several to many different RNP1s are preassembledas described above in Example II with gRNA sequences designed to 1)query Y and/or X chromosome loci in sex mismatch transplantation cases;or 2) gRNA sequences designed to query various loci that are differentin the genomic DNA of the recipient and the donor; along with anLbCas12a nuclease (i.e., an DNA-specific nuclease). Also as described inExample II above, an RNP2 is preassembled with a gRNA sequence designedto target an unblocked nucleic acid molecule that results fromunblocking (i.e., linearizing) a chosen blocked nucleic acid moleculesuch as U29. The blocked nucleic acid molecule is formed as describedabove in Example III, and a reporter is formed as described above inExample IV. The reaction mix contains the preassembled RNP1,preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pHof about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay isperformed by one of the protocols described above in Example V. Thereadout detects the level of donor-specific nucleic acid sequences.

Example XIII: Detection of Microbe Contamination in a Laboratory

DNA that is found in the environment is called “environmental DNA” oreDNA (e-DNA) for short, and it is formally defined as “genetic materialobtained directly from environmental samples without any obvious signsof biological source material.” eDNA has been harnessed to detect rareor invasive species and pathogens in a broad range of environments.Samples are typically collected in the form of water, soil, sediment, orsurface swabs. The DNA must then be extracted and purified to removechemicals that may inhibit the cascade reaction. Surface wipe samplesare commonly collected to assess microbe contamination in, e.g., alaboratory. The wipe test protocol consists of four distinct stages:removal of DNA from surfaces using absorbent wipes, extraction of DNAfrom the wipes into a buffer solution, purification of DNA, and analysisof the extract.

For sample collection, sterile 2×2 inch polyester-rayon non-woven wipesare used to wipe down an environmental surface, such as a laboratorybench. Each wipe is placed into a sterile 50 ml conical tube and 10 mLof PBST is transferred to each conical tube using a sterile serologicalpipette. The tubes are vortexed at the maximum speed for 20 minutesusing a Vortex Genie 2. A 200 μL aliquot of the supernatant wasprocessed using a nucleic acid purification kit (QIAmp DNA Blood MiniKit, QIAGEN, Inc., Valencia, CA). The kit lyses the sample, stabilizesand binds DNA to a selective membrane, and elutes the DNA sample.

For the cascade assay, several to many different RNP1s are preassembledas described above in Example II with gRNA sequences designed to detect,e.g., Aspergillus acidus; Parafilaria bovicola; Babesia divergens;Escherichia coli; Pseudomonas aeruginosa; and Dengue virus; along withan LbCas12a nuclease (i.e., an DNA-specific nuclease). Also as describedin Example II above, an RNP2 is preassembled with a gRNA sequencedesigned to target an unblocked nucleic acid molecule that results fromunblocking (i.e., linearizing) a chosen blocked nucleic acid moleculesuch as U29. The blocked nucleic acid molecule is formed as describedabove in Example III, and a reporter is formed as described above inExample IV. The reaction mix contains the preassembled RNP1,preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pHof about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay isperformed by one of the protocols described above in Example V. Thereadout is detection of a genomic sequence unique to a pathogen.

While certain embodiments have been described, these embodiments havebeen presented by way of example only and are not intended to limit thescope of the present disclosures. Indeed, the novel methods,apparatuses, modules, instruments and systems described herein can beembodied in a variety of other forms; furthermore, various omissions,substitutions and changes in the form of the methods, apparatuses,modules, instruments and systems described herein can be made withoutdeparting from the spirit of the present disclosures. The accompanyingclaims and their equivalents are intended to cover such forms ormodifications as would fall within the scope and spirit of the presentdisclosures.

We claim:
 1. A method for preventing unwinding of blocked nucleic acidmolecules in the presence of an RNP comprising the steps of: providingblocked nucleic acid molecules; providing ribonucleoprotein complexescomprising a Cas12a nucleic acid-guided nuclease that exhibits both cis-and trans-cleavage activity upon activation and a gRNA that recognizesan unblocked nucleic acid molecule resulting from trans-cleavage of theblocked nucleic acid molecules; and engineering the Cas12a nucleicacid-guided nuclease to comprise at least one mutation to domains thatinteract with the PAM region or surrounding sequences on the blockednucleic acid molecule to result in a variant nucleic acid-guidednuclease where single stranded DNA is cleaved faster than doublestranded DNA is cleaved.
 2. The method of claim 1, wherein the blockednucleic acid molecules comprise a structure represented by any one ofFormulas I-IV, wherein Formulas I-IV are in the 5′-to-3′ direction:(a) A-(B-L)_(J)-C-M-T-D  (Formula I); wherein A is 0-15 nucleotides inlength; B is 4-12 nucleotides in length; L is 3-25 nucleotides inlength; J is an integer between 1 and 10; C is 4-15 nucleotides inlength; M is 1-25 nucleotides in length or is absent, wherein if M isabsent then A-(B-L) J-C and T-D are separate nucleic acid strands; T is17-135 nucleotides in length and comprises at least 50% sequencecomplementarity to B and C; and D is 0-10 nucleotides in length andcomprises at least 50% sequence complementarity to A;(b) D-T-T′-C-(L-B)_(J)-A  (Formula II); wherein D is 0-10 nucleotides inlength; T-T′ is 17-135 nucleotides in length; T′ is 1-10 nucleotides inlength and does not hybridize with T; C is 4-15 nucleotides in lengthand comprises at least 50% sequence complementarity to T; L is 3-25nucleotides in length and does not hybridize with T; B is 4-12nucleotides in length and comprises at least 50% sequencecomplementarity to T; J is an integer between 1 and 10; A is 0-15nucleotides in length and comprises at least 50% sequencecomplementarity to D;(c) T-D-M-A-(B-L)_(J)-C  (Formula III); wherein T is 17-135 nucleotidesin length; D is 0-10 nucleotides in length; M is 1-25 nucleotides inlength or is absent, wherein if M is absent then T-D and A-(B-L)_(J)-Care separate nucleic acid strands; A is 0-15 nucleotides in length andcomprises at least 50% sequence complementarity to D; B is 4-12nucleotides in length and comprises at least 50% sequencecomplementarity to T; L is 3-25 nucleotides in length; J is an integerbetween 1 and 10; and C is 4-15 nucleotides in length; or(d) T-D-M-A-L_(p)-C  (Formula IV); wherein T is 17-31 nucleotides inlength (e.g., 17-100, 17-50, or 17-25); D is 0-15 nucleotides in length;M is 1-25 nucleotides in length; A is 0-15 nucleotides in length andcomprises a sequence complementary to D; and L is 3-25 nucleotides inlength; p is 0 or 1; C is 4-15 nucleotides in length and comprises asequence complementary to T.
 3. The method of claim 3, wherein: (a) T ofFormula I comprises at least 80% sequence complementarity to B and C;(b) D of Formula I comprises at least 80% sequence complementarity to A;(c) C of Formula II comprises at least 80% sequence complementarity toT; (d) B of Formula II comprises at least 80% sequence complementarityto T; (e) A of Formula II comprises at least 80% sequencecomplementarity to D; (f) A of Formula III comprises at least 80%sequence complementarity to D; (g) B of Formular III comprises at least80% sequence complementarity to T; (h) A of Formula IV comprises atleast 80% sequence complementarity to D; and/or (i) C of Formula IVcomprises at least 80% sequence complementarity to T.
 4. The method ofclaim 1, wherein the Cas12a nucleic acid-guided nuclease comprises amutation selected from mutations to amino acid residues K548, N552 andK607 in relation to SEQ ID NO:2.
 5. The method of claim 4, wherein theCas12a nucleic acid-guided nuclease comprises at least two mutationsselected from mutations to amino acid residues K548, N552 and K607 inrelation to SEQ ID NO:2.
 6. The method of claim 5, wherein the Cas12anucleic acid-guided nuclease comprises mutations to amino acid residuesK548, N552 and K607 in relation to SEQ ID NO:2.
 7. The method of claim1, wherein the Cas12a nucleic acid-guided nuclease comprises a mutationselected from mutations to amino acid residues K534, Y538 and R591 inrelation to SEQ ID NO:3.
 8. The method of claim 7, wherein the Cas12anucleic acid-guided nuclease comprises at least two mutations selectedfrom mutations to amino acid residues K534, Y538 and R591 in relation toSEQ ID NO:3.
 9. The method of claim 8, wherein the Cas12a nucleicacid-guided nuclease comprises mutations to amino acid residues K534,Y538 and R591 in relation to SEQ ID NO:3.
 10. The method of claim 1,wherein the Cas12a nucleic acid-guided nuclease comprises a mutationselected from mutations to amino acid residues K541, N545 and K601 inrelation to SEQ ID NO:4.
 11. The method of claim 10, wherein the Cas12anucleic acid-guided nuclease comprises at least two mutations selectedfrom mutations to amino acid residues K541, N545 and K601 in relation toSEQ ID NO:4.
 12. The method of claim 11, wherein the Cas12a nucleicacid-guided nuclease comprises mutations to amino acid residues K541,N545 and K601 in relation to SEQ ID NO:4.
 13. The method of claim 1,wherein the Cas12a nucleic acid-guided nuclease comprises a mutationselected from mutations to amino acid residues K579, N583 and K635 inrelation to SEQ ID NO:5.
 14. The method of claim 13, wherein the Cas12anucleic acid-guided nuclease comprises at least two mutations selectedfrom mutations to amino acid residues K579, N583 and K635 in relation toSEQ ID NO:5.
 15. The method of claim 14, wherein the Cas12a nucleicacid-guided nuclease comprises mutations to amino acid residues K579,N583 and K635 in relation to SEQ ID NO:5.
 17. The method of claim 1,wherein the Cas12a nucleic acid-guided nuclease comprises a mutationselected from mutations to amino acid residues K613, N617 and K671 inrelation to SEQ ID NO:6.
 18. The method of claim 17, wherein the Cas12anucleic acid-guided nuclease comprises at least two mutations selectedfrom mutations to amino acid residues K613, N617 and K671 in relation toSEQ ID NO:6.
 19. The method of claim 18, wherein the Cas12a nucleicacid-guided nuclease comprises mutations to amino acid residues K613,N617 and K671 in relation to SEQ ID NO:6.
 20. The method of claim 1,wherein the Cas12a nucleic acid-guided nuclease comprises a mutationselected from mutations to amino acid residues K613, N617 and K671 inrelation to SEQ ID NO:7.
 21. The method of claim 20, wherein the Cas12anucleic acid-guided nuclease comprises at least two mutations selectedfrom mutations to amino acid residues K613, N617 and K671 in relation toSEQ ID NO:7.
 22. The method of claim 21, wherein the Cas12a nucleicacid-guided nuclease comprises mutations to amino acid residues K613,N617 and K671 in relation to SEQ ID NO:7.
 23. The method of claim 1,wherein the Cas12a nucleic acid-guided nuclease comprises a mutationselected from mutations to amino acid residues K617, N621 and K678 inrelation to SEQ ID NO:8.
 24. The method of claim 23, wherein the Cas12anucleic acid-guided nuclease comprises at least two mutations selectedfrom mutations to amino acid residues K617, N621 and K678 in relation toSEQ ID NO:8.
 25. The method of claim 24, wherein the Cas12a nucleicacid-guided nuclease comprises mutations to amino acid residues K617,N621 and K678 in relation to SEQ ID NO:8.
 26. The method of claim 1,further comprising the steps of: providing a sample putativelycomprising a target nucleic acid of interest; and providing secondribonucleoprotein complexes comprising a second nucleic acid-guidednuclease that exhibits both cis- and trans-cleavage activity uponactivation and a second gRNA that recognizes the target nucleic acid ofinterest.
 27. The method of claim 26, wherein the target nucleic acid ofinterest is a DNA nucleic acid and the second nucleic acid-guidednuclease is a Cas12a or Cas14a.
 28. The method of claim 26, wherein thetarget nucleic acid of interest is an RNA nucleic acid and the secondnucleic acid-guided nuclease is a Cas12g or Cas13a.
 29. The method ofclaim 26, further comprising the step of providing reporter moieties.30. The method of claim 29, wherein the reporter moieties comprise aFRET pair.